Lysosomal Ca2+ as a mediator of palmitate-induced lipotoxicity

Soo-Jin Oh; Yeseong Hwang; Kyu Yeon Hur; Myung-Shik Lee

doi:10.1038/s41420-023-01379-0

Lysosomal Ca2+ as a mediator of palmitate-induced lipotoxicity

Oh, Soo-Jin; Hwang, Yeseong; Hur, Kyu Yeon; Lee, Myung-Shik 2023-03-21 00:00:00 www.nature.com/cddiscovery ARTICLE OPEN 2+ Lysosomal Ca as a mediator of palmitate-induced lipotoxicity 1,2,5 3,5 4 2,3 Soo-Jin Oh , Yeseong Hwang , Kyu Yeon Hur and Myung-Shik Lee © The Author(s) 2023 While the mechanism of lipotoxicity by palmitic acid (PA), an effector of metabolic stress in vitro and in vivo, has been extensively investigated, molecular details of lipotoxicity are still not fully characterized. Since recent studies reported that PA can exert lysosomal stress in addition to well-known ER and mitochondrial stress, we studied the role of lysosomal events in lipotoxicity by 2+ PA, focusing on lysosomal Ca . We found that PA induced accumulation of mitochondrial ROS and that mitochondrial ROS 2+ 2+ 2+ induced release of lysosomal Ca due to lysosomal Ca exit channel activation. Lysosomal Ca release led to increased cytosolic 2+ 2+ Ca which induced mitochondrial permeability transition (mPT). Chelation of cytoplasmic Ca or blockade of mPT with olesoxime 2+ 2+ or decylubiquinone (DUB) suppressed lipotoxicity. Lysosomal Ca release led to reduced lysosomal Ca content which was 2+ 2+ 2+ replenished by ER Ca , the largest intracellular Ca reservoir (ER→ lysosome Ca reﬁlling), which in turn activated store- 2+ 2+ 2+ operated Ca entry (SOCE). Inhibition of ER→ lysosome Ca reﬁlling by blockade of ER Ca exit channel using dantrolene or inhibition of SOCE using BTP2 inhibited lipotoxicity in vitro. Dantrolene or DUB also inhibited lipotoxic death of hepatocytes in vivo induced by administration of ethyl palmitate together with LPS. These results suggest a novel pathway of lipotoxicity characterized 2+ 2+ by mPT due to lysosomal Ca release which was supplemented by ER→ lysosome Ca reﬁlling and subsequent SOCE, and also 2+ 2+ suggest the potential role of modulation of ER→ lysosome Ca reﬁlling by dantrolene or other blockers of ER Ca exit channels in disease conditions characterized by lipotoxicity such as metabolic syndrome, diabetes, cardiomyopathy or nonalcoholic steatohepatitis. Cell Death Discovery (2023) 9:100 ; https://doi.org/10.1038/s41420-023-01379-0 INTRODUCTION RESULTS 2+ Palmitic acid (PA) is an important molecule acting as an effector PA elicits lysosomal Ca release of lipid injury and metabolic stress in vitro and in vivo. PA can induce reactive oxygen species (ROS) by compromising Mechanisms of PA-induced lipotoxicity have been ascribed to mitochondrial complex I and III [11, 16], which can activate lysosomal 2+ 2+ 2+ endoplasmic reticulum (ER) stress, mitochondrial stress, pro- Ca channel [12, 17]. Lysosomal Ca release due to lysosomal Ca 2+ 2+ duction of ceramide or lysophosphatidylcholine [1–4], leading exit channel activation can increase cytosolic Ca content ([Ca ] ), to JNK activation, apoptosis or necrosis, depending on the which, in turn, can induce mitophagy, lysosomal stress response experimental condition and cellular context [3, 5–7]. While ER [11, 12] or diverse types of cell death [18–20]. Hence, we studied and mitochondria are well-established target organelles of PA- whether PA-induced lipotoxicity entails mitochondrial ROS-induced 2+ 2+ induced injury [2, 4, 8, 9], lysosomal changes have recently been lysosomal Ca release and increased [Ca ] . reported to occur such as decreased lysosomal acidity, altered When we treated HepG2 cells with PA, signiﬁcant lipotoxic cell lysosomal enzyme activity or impaired lysosomal integrity death was observed in a dose range of 500 ~ 1,000 μM, as 2+ [10–13]. We recently reported that lysosomal Ca is released revealed by SYTOX Green staining (Fig. 1A). Furthermore, after treatment with PA, while the cellular consequences of accumulation of mitochondrial ROS stained with MitoSOX was 2+ lysosomal Ca releasecouldbedistinctdepending on thecell well visualized in the same dose range of PA, which was types or context of treatment [11, 12]. Since release of signiﬁcantly reduced by MitoTEMPO, a mitochondrial ROS 2+ 2+ lysosomal Ca can also lead to increased cytosolic Ca quencher (Fig. 1B), indicating mitochondrial ROS generation by 2+ 2+ content ([Ca ] )[11, 12]and Ca is one of the most important PA. As PA doses higher than 500 μM could be unphysiologically inducers of mitochondrial permeability transition (mPT) and high [21], we employed 500 μM of PA in the following subsequent cell death [14, 15], we conducted this investigation experiments to minimize unphysiological effects of high dose of 2+ based on our hypothesis that lysosomal Ca release by PA can PA. In an experiment to ﬁnd causal relationship between lipotoxic lead to lipotoxic cell death through mPT. cell death and mitochondrial ROS, PA-induced death of HepG2 1 2 Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul 06355, Korea. Department of Integrated Biomedical Science, Soonchunhyang Institute of Medi-bio Science and Division of Endocrinology, Department of Internal Medicine, Soonchunhyang Medical Center, Soonchunhyang University College of Medicine, Cheonan, Korea. Severance Biomedical Science Institute, Graduate school of Medical Science, BK21 Project, Yonsei University College of Medicine, Seoul 03722, Korea. 4 5 Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. These authors contributed equally: Soo-Jin Oh, Yeseong Hwang. email: mslee0923@sch.ac.kr Edited by Professor Myung-Shik Lee Received: 4 September 2022 Revised: 22 February 2023 Accepted: 23 February 2023 Ofﬁcial journal of CDDpress 1234567890();,: S.-J. Oh et al. cells was signiﬁcantly reduced by MitoTEMPO (Fig. 1C), suggesting stained with Oregon Green-488 BAPTA-1 dextran (OGBD) was that mitochondrial ROS generation is a critical event in PA-induced signiﬁcantly decreased in HepG2 cells treated with PA (Fig. 1D), 2+ 2+ lipotoxicity. When we studied possible changes of lysosomal Ca suggesting release of lysosomal Ca by PA treatment, similar to 2+ 2+ after PA treatment, concentration of lysosomal Ca ([Ca ] ) the results using other types of cells [11, 12]. When we indirectly Lys Cell Death Discovery (2023) 9:100 S.-J. Oh et al. 2+ Fig. 1 PA reduces lysosomal Ca .A After treatment of HepG2 cells with 250–1000 μM PA for 24 h, cell death was determined using SYTOX Green. B After treatment of HepG2 cells with 250–1000 μM PA for 24 h in the presence or absence of 10 μM MitoTEMPO, PA-induced mitochondrial ROS was measured by MitoSOX followed by ﬂow cytometry (lower panel). Representative scattergrams are shown (upper panel). C After treatment of HepG2 cells with 500 μM PA for 24 h in the presence or absence of 10 μM MitoTEMPO, cell death was determined using SYTOX Green. D Cells were loaded with Oregon Green-488 BAPTA-1 Dextran (OGBD) for 16 h and chased for 4 h. Cells were then treated 2+ with PA or BSA for 6 h in the presence or absence of 10 μM MitoTEMPO, followed by confocal microscopy to determine [Ca ] (right). Lys Representative ﬂuorescence images are shown (left panel). Scale bar, 20 μm. E After staining with Fluo-3 AM for 30 min, cells were treated in 2+ 2+ PA or BSA for 6 h, followed by confocal microscopy to monitor GPN-induced changes of lysosomal Ca release and [Ca ] increase (left). AUC 2+ of the curve was calculated as an estimate of lysosomal Ca content (right). F Cells transfected with GCaMP3-ML1 were treated with PA or BSA 2+ for 6 h, and then GPN-induced change of GCaMP3-ML1 ﬂuorescence was determined by confocal microscopy to visualize perilysosomal Ca 2+ release (right). Representative ﬂuorescence images are shown (left panel). Scale bar, 10 μm. G [Ca ] in HepG2 cells treated with PA for 6 h was determined by staining with Fluo-3 AM (middle) or ratiometric analysis after Fura-2 loading (right). Representative Fluo-3 ﬂuorescence images 2+ are shown (left panel). Scale bar, 20 μm. H [Ca ] in HepG2 cells treated with PA for 6 h in the presence or absence of 10 μM MitoTEMPO was determined by ratiometric analysis after Fura-2 loading. I After treatment of HepG2 cells with PA in the presence or absence of 10 μM CA- 074Me for 24 h, cell death was determined using SYTOX Green. Data are expressed as the means ± SD of three independent experiments. Statistical comparisons were performed using one-way ANOVA with Tukey’s multiple comparison test (A–D, H, I) or two-tailed unpaired Student’s t-test (vehicle and PA comparisons in E–G). (*P < 0.05; **P < 0.01; ***P < 0.001; ns not signiﬁcant). 2+ estimated lysosomal Ca content by calculating area under the can lead to cell death such as necrosis or apoptosis depending on 2+ 2+ curve (AUC) of cytosolic Ca concentration ([Ca ] ) tracing using the severity of mPT and cellular context [15, 27, 28], we studied Fluo-3 AM staining after treatment with Gly-Phe β-naphthylamide the role of mPT in HepG2 cell death by PA using inhibitors of mPT. 2+ (GPN), a lysosomotropic agent [22], lysosomal Ca content was Indeed, mPT inhibitors such as olesoxime [29] or decylubiquinone again signiﬁcantly reduced by PA treatment (Fig. 1E). Decrease of (DUB) [14], signiﬁcantly reduced PA-induced cell death assessed 2+ [Ca ] was abrogated by MitoTEMPO (Fig. 1D), suggesting that by SYTOX Green staining or LDH release assay (Fig. 2B, C). Reduced Lys mitochondrial ROS produced by PA treatment induces lysosomal lipotoxic cell death by olesoxime or DUB was accompanied by 2+ 2+ 2+ Ca release. We also studied whether perilysosomal Ca release signiﬁcantly reduced mPT as evidenced by decreased Co can be observed after PA treatment employing GCaMP3-ML1, a quenching of calcein ﬂuorescence (Fig. 2D, E), indicating that 2+ 2+ probe detecting perilysosomal Ca release [22]. In GCaMP3-ML1- Ca -mediated mPTP opening is an important mechanism of PA- 2+ transfected HepG2 cells, perilysosomal Ca release was not induced lipotoxicity. PA-induced HepG2 cell death was also directly visualized by PA treatment (Fig. 1F). However, perilysoso- signiﬁcantly reduced by BAPTA-AM, conﬁrming the role of 2+ 2+ 2+ mal Ca release after treatment with GPN was markedly reduced increased [Ca ] and Ca -mediated mPTP in lipotoxicity of 2+ (Fig. 1F), suggesting pre-emptying or release of lysosomal Ca HepG2 cells (Fig. 2F). As a consequence of mitochondrial damage after PA treatment, similar to the results using other types of cells associated with mPT, mitochondrial potential determined by [12, 17]. TMRE staining was signiﬁcantly lowered by PA treatment of 2+ When we determined [Ca ] and studied whether release of HepG2 cells (Fig. 2G). 2+ 2+ 2+ lysosomal Ca leads to increased cytosolic Ca content ([Ca ] ), 2+ [Ca ] estimated by Fluo-3 AM staining was signiﬁcantly ER→ lysosome calcium ﬂux and SOCE in lipotoxicity 2+ 2+ increased by PA (Fig. 1G), supporting release of lysosomal Ca While we showed the role of lysosomal Ca release in lipotoxicity, 2+ 2+ 2+ to the cytosol. When ratiometric measurement of [Ca ] after lysosomal Ca might not be a sufﬁcient source of Ca required 2+ Fura-2 staining was conducted to avoid interference due to for full execution of cellular process requiring Ca , since 2+ 2+ uneven loading or photobleaching [23], increased [Ca ] by PA lysosomal volume is 1% of cell volume and lysosomal Ca pool 2+ treatment was again well observed, which was reversed by is a relatively small Ca reservoir [30]. We thus wondered 2+ MitoTEMPO (Fig. 1G, H), indicating that mitochondrial ROS- whether release or emptying of lysosomal Ca content in HepG2 2+ 2+ induced lysosomal Ca release leads to increased [Ca ] . Since cells treated with PA could be replenished from endoplasmic 2+ 2+ molecules other than Ca might be released from lysosome and reticulum (ER), the largest intracellular Ca reservoir [31] which 2+ could affect cell viability, we studied effect of an inhibitor of has been observed when lysosomal Ca emptying occurs (i.e., 2+ 2+ cathepsin B that has been reported to be released from ER→ lysosome Ca reﬁlling) [12, 31]. When [Ca ] was ER hepatocytes after PA treatment and to induce lipotoxic cell death determined after PA treatment of HepG2 cells using GEM- 2+ [24]. Ca-074Me, a cell-permeable inhibitor of cathepsin B, did not CEPIA1er [32]in a Ca -free Krebs-Ringer bicarbonate (KRB) buffer 2+ 2+ inhibit cell death by PA, suggesting no role of lysosomal cathepsin to abolish possible store-operated Ca entry (SOCE) [33], Ca 2+ B release in lipotoxic cell death of HepG2 cells (Fig. 1I). Taken concentration in the ER ([Ca ] ) was signiﬁcantly reduced (Fig. ER 2+ together, these data demonstrate that PA-induced mitochondrial 3A), suggesting that ER Ca is mobilized probably to replenish 2+ 2+ 2+ ROS leads to release of lysosomal Ca to cytoplasm and lysosomal Ca loss. When [Ca ] was determined without ER 2+ 2+ 2+ increased [Ca ] , contributing to lipotoxic HepG2 cell death. removal of extracellular Ca , [Ca ] was not signiﬁcantly i ER 2+ reduced likely due to activation of store-operated Ca entry 2+ 2+ mPT pore (mPTP) opening mediates lysosomal Ca loss- (SOCE) after ER Ca emptying (Fig. 3A), which suggests that driven cell death in response to PA SERCA was not inhibited by PA since SERCA inhibition reduces 2+ 2+ 2+ We next studied whether PA-induced release of Ca from [Ca ] regardless of extracellular Ca [32] and eliminates the ER 2+ 2+ lysosome into cytosol can induce mPTP opening because Ca is possibility of increased [Ca ] after PA treatment due to SERCA one of the most important inducers of mPT which can lead to inhibition. 2+ mitochondrial catastrophe and cell death [2, 25]. When cells were Since these results suggested replenishment of lysosomal Ca 2+ stained by calcein-AM/CoCl staining, mPTP opening visualized by depletion by ER Ca , we next studied whether ER to lysosomal 2+ 2+ Co quenching of mitochondrial matrix calcein ﬂuorescence [26] Ca movement indeed occurs after PA treatment inducing 2+ was well detected after PA treatment of HepG2 cells, showing lysosomal Ca release. When PA was removed after treatment for 2+ occurrence of mPT by PA (Fig. 2A). PA-induced mPT was 6 h, a decrease of [Ca ] by PA treatment was recovered (Fig. Lys 2+ 2+ abrogated by BAPTA-AM chelating intracellular Ca (Fig. 2A), 3B). To study the role of ER Ca in the recovery of reduced 2+ 2+ 2+ indicating Ca -dependent mPT after PA treatment. Since mPT [Ca ] , we studied the effect of blockade of ER Ca exit Lys Cell Death Discovery (2023) 9:100 S.-J. Oh et al. 2+ channels that could be routes of ER to lysosomal Ca movement hand, dantrolene, an antagonist of ryanodine receptor (RyR), 2+ 2+ during the recovery of [Ca ] . When we employed Xestospon- another ER Ca exit channel, signiﬁcantly suppressed the Lys 2+ 2+ gin C, an IP3R antagonist, recovery of decreased [Ca ] after recovery of suppressed [Ca ] after removal of PA (Fig. 3B), Lys Lys 2+ removal of PA was not signiﬁcantly affected (Fig. 3B). On the other suggesting involvement of RyR channel in ER→ lysosome Ca Cell Death Discovery (2023) 9:100 S.-J. Oh et al. 2+ Fig. 2 mPTP opening by PA due to lysosomal Ca release. A After treatment of HepG2 cells with 500 μM PA in the presence or absence of 2+ 30 μM BAPTA-AM for 24 h, mPTP opening was assessed by ﬂow cytometry after calcein-AM loading and Co quenching (right). Representative histograms are shown (left). B, C After treatment of HepG2 cells with PA in the presence or absence of 100 μM olesoxime (B)or 200 μM DUB (C) for 24 h, cell death was determined using SYTOX Green (left) or LDH release assay (right). D, E After treatment of HepG2 cells with PA in the presence or absence of 100 μM olesoxime (D) or 200 μM DUB (E) for 24 h, mPTP opening was determined as in (A) (right). Representative histograms are shown (left). F After the same treatment as in (A), cell death was determined using SYTOX Green (left) or LDH release assay (right). G After treatment of HepG2 cells with PA as in (A), mitochondrial potential was determined by TMRE staining (right). Representative ﬂuorescence images are shown (left). Scale bar, 20 μm. Data are expressed as the means ± SD of three independent experiments. All statistical comparisons were performed using one-way ANOVA with Tukey’s multiple comparison test. (*P < 0.05; **P < 0.01; ***P < 0.001; ns not signiﬁcant). 2+ reﬁlling after lysosome Ca release by PA. When we chelated ER by PA determined by Fluo-3 AM staining or ratiometric measure- 2+ Ca with a membrane-permeant metal chelator N,N,N’,N’-tetrakis ment after Fura-2 loading was signiﬁcantly attenuated by BTP2 2+ 2+ (2-pyridylmethyl) ethylene diamine (TPEN) that has a low Ca (Fig. 4C, D), indicating the role of SOCE in the increase of [Ca ] by 2+ 2+ afﬁnity and can chelate ER Ca but not cytosolic Ca [34], PA. Furthermore, PA-induced death of HepG2 cells was signiﬁ- 2+ recovery of [Ca ] after removal of PA was signiﬁcantly cantly reduced by BTP2 (Fig. 4E), supporting the role of SOCE in Lys 2+ 2+ 2+ inhibited (Fig. 3B), again supporting the role of ER Ca in the Ca -mediated lipotoxicity. EGTA chelating extracellular Ca also 2+ 2+ recovery of lysosomal Ca . signiﬁcantly reduced increase of [Ca ] and death of HepG2 cells 2+ We next studied the effect of blockade of ER Ca exit channels after PA treatment (Fig. 4C, F), further supporting the role of 2+ 2+ 2+ on the increase of [Ca ] after PA treatment. Again, dantrolene extracellular Ca ﬂux in the increase of [Ca ] and subsequent i i but not Xestospongin C, signiﬁcantly suppressed the increase of lipotoxicity. 2+ [Ca ] after PA treatment determined by ratiometric measure- ment following Fura-2 loading (Fig. 3C), suggesting that ER→ In vivo lipotoxicity 2+ 2+ lysosome Ca reﬁlling through RyR channel contributes to the Based on our in vitro results showing the role of lysosomal Ca 2+ 2+ increase of [Ca ] after PA treatment by replenishing decreased release coupled with ER→ lysosome Ca reﬁlling in mPT and 2+ 2+ lysosomal Ca content and sustaining lysosomal Ca release. cell death in vitro, we next studied the effect of blockade of 2+ 2+ TPEN that reduced recovery of decreased [Ca ] also attenuated lysosomal Ca -mediated mPT on lipotoxicity in vivo. When Lys 2+ increase of [Ca ] after PA treatment (Fig. 3C). When cell death mice were injected with ethyl palmitate followed by LPS was determined, dantrolene but not Xestospongin C, signiﬁcantly administration, death of hepatocytes was observed by TUNEL suppressed the cell death after PA treatment (Fig. 3D), suggesting staining which was accompanied by elevated serum alanine 2+ that ER→ lysosome Ca reﬁlling through RyR channel contributes aminotransferase (ALT)/aspartate aminotransferase (AST) (Fig. to the PA-induced cell death probably by supporting continuous 5A, B), similar to a previous paper [37]. When dantrolene that 2+ increase of cytosolic Ca and subsequent mPTP opening. TPEN was able to inhibit PA-induced hepatocyte death through 2+ also alleviated HepG2 cell death by PA (Fig. 3D), substantiating blockade of the increase of [Ca ] was administered to mice 2+ supportive role of ER Ca in lipotoxicity. To study dynamic before ethyl palmitate injection, death of hepatocytes identiﬁed 2+ changes of [Ca ] and its temporal relationship with lysosomal by TUNEL staining was signiﬁcantly ameliorated, suggesting ER 2+ 2+ 2+ Ca reﬁlling, we simultaneously traced [Ca ] and [Ca ] in blockade of in vivo lipotoxicity by dantrolene (Fig. 5A). Elevated ER Lys cells transfected with GEM-CEPIA1er and loaded with OGBD. When serum ALT/AST levels after ethyl palmitate followed by LPS 2+ organelle [Ca ] was monitored in cells that have reduced administration were also signiﬁcantly reduced by dantrolene 2+ [Ca ] after PA treatment for 6 h and subsequently were pretreatment (Fig. 5B). In addition, accumulation of mitochon- Lys 2+ 2+ incubated in a Ca -free medium, a decrease of [Ca ] occurred drial ROS stained by MitoSOX was observed in the liver tissue of ER 2+ in parallel with an increase of [Ca ] (Fig. 3E), which strongly mice treated with LPS + ethyl palmitate (Fig. 5C), which is in line Lys 2+ supports that ER→ lysosome Ca reﬁlling occurs during HepG2 with mitochondrial ROS accumulation in HepG2 cells treated cell lipotoxicity. with PA in vitro. Such mitochondrial ROS accumulation in vivo 2+ Decrease of [Ca ] after PA treatment only in the absence of was ameliorated by dantrolene administration (Fig. 5C). We also ER 2+ extracellular Ca suggested SOCE without SERCA inhibition studied whether blockade of mPT could inhibit lipotoxicity 2+ because SERCA inhibition would decrease [Ca ] regardless of in vivo based on our in vitro results showing the role of mPT in ER 2+ extracellular Ca [32]. Thus, we next studied possible occurrence PA-induced lipotoxicity. When we pretreated mice with DUB, an and role of SOCE in lipotoxicity by PA. Since ER luminal protein inhibitor of mPT, hepatocyte death detected by TUNEL staining STIM1 oligomerizes and recruits plasma membrane SOCE channel or elevated serum ALT/AST levels was signiﬁcantly reduced (Fig. 2+ 2+ ORAI1 to activate Ca entry when ER Ca stores are reduced 5A, B), indicating the role of mPT in lipotoxicity in vivo. [33], we studied expression pattern of STIM1 after PA treatment Mitochondrial ROS in the liver tissue of mice treated with 2+ 2+ that reduced ER Ca store due to ER→ lysosome Ca reﬁlling. In LPS + ethyl palmitate was also reduced by DUB (Fig. 5C). These control-treated cells, neither STIM1 oligomerization nor co- results showing amelioration of PA-induced hepatocyte death localization between STIM1 and ORAI1 was observed (Fig. 4A). In in vivo by dantrolene or DUB, suggestthatlipotoxicityof 2+ contrast, both STIM1 oligomerization and co-localization between hepatocytes in vivo can be mediated by Ca -mediated mPT 2+ STIM1 and ORAI1 were well observed after PA treatment (Fig. 4A), and indicate the role of lysosomal Ca release supported by 2+ 2+ supporting that reduced ER Ca content after PA treatment ER→ lysosome Ca reﬁlling and SOCE in mPT-induced lipo- induced SOCE activation. To validate the role of SOCE in toxicity observed in vitro. 2+ intracellular Ca ﬂux, we studied the effect of BTP2, a blocker 2+ of SOCE [35, 36] on [Ca ] . Indeed, in the presence of BTP2, ER 2+ [Ca ] was signiﬁcantly reduced by PA even without removal of DISCUSSION ER 2+ extracellular Ca . In contrast, without BTP2, PA-induced reduction It is well established that PA, as an effector of metabolic stress 2+ 2+ of [Ca ] was not observed in the presence of extracellular Ca in vitro, can induce stress in diverse organelles such as ER and ER (Fig. 4B), suggesting the occurrence of SOCE after PA treatment mitochondria [2, 4, 8, 9]. However, recent investigation showed 2+ 2+ likely due to ER→ lysosome Ca reﬁlling and decreased [Ca ] that PA can induce stress or dysfunction of lysosome as well ER 2+ 2+ unrelated to SERCA inhibition. Furthermore, the increase of [Ca ] [10–13], such as elevated pH or reduced Ca content due to Cell Death Discovery (2023) 9:100 S.-J. Oh et al. 2+ Fig. 3 Role of ER→ lysosome Ca reﬁlling in lipotoxicity. A After treatment of GEM-CEPIA1er-transfected HepG2 cells with PA for 6 h in a 2+ 2+ Ca -free KRBB (right) or a full medium (left), [Ca ] was determined by confocal microscopy. B After treatment of OGBD-labeled cells with ER PA for 6 h, cells were incubated in a fresh medium without PA in the presence or absence of 10 μM dantrolene (Dan), 10 μM TPEN or 3 μM 2+ Xestospongin C (Xesto C). Recovery of [Ca ] after removal of PA was determined by confocal microscopy (lower). Representative Lys 2+ ﬂuorescence images are shown (upper). Scale bar, 20 μm. C After the same treatment as in B, [Ca ] was determined by radiometric analysis after Fura-2 loading. D After the same treatment as in (B), cell death was evaluated by SYTOX Green staining. E After treatment of GEM- 2+ 2+ CEPIA1er transfected cells loaded with OGBD with PA for 6 h and washout, recovery of [Ca ] and changes of [Ca ] were monitored Lys ER 2+ simultaneously in the absence of extracellular Ca . Data are expressed as the means ± SD of three independent experiments. Statistical comparisons were performed using two-tailed Student’s t-test (A) or one-way ANOVA with Tukey’s multiple comparison test (B–D). (*P < 0.05; **P < 0.01; ***P < 0.001; ns not signiﬁcant). 2+ 2+ 2+ release of Ca from lysosome. Increased cytosolic Ca can channel responsible for PA-induced lysosomal Ca release is not 2+ mediate diverse beneﬁcial or harmful effects on cells. Since Ca is clear. Previous papers have reported the role of TRPML1 or TRPM2 2+ 2+ a well-known inducer of mPT [14, 15], we hypothesized that channel in Ca -orZn -mediated cell death [39–41]. We have 2+ lysosomal Ca release might contribute to cell death after also studied the effect of inhibitors of TRPML1 or TRPM2. However, 2+ treatment with PA or lipotoxicity. Indeed, we observed that Ca ML-SI1, ML-SI3 or N-(p-amylcinnamoyl)anthranilic acid inhibiting could be released from lysosome after PA treatment, which is TRPML1, TRPML1/2/3 and TRPM2, respectively [42–44], did not 2+ likely due to activation of lysosomal Ca exit channel by inhibit PA-induced lipotoxic death of HepG2 cells (Oh S-J et al., 2+ 2+ mitochondrial ROS. After release of lysosomal Ca , [Ca ] was unpublished results). Furthermore, Ned-19, an inhibitor of TPC1/2 increased, which imposed mPT and subsequent death. Cell death which has been reported to be associated with ischemia- 2+ due to mPT caused by PA-induced lysosomal Ca release was reperfusion injury [45], was also without effect (Oh S-J et al., 2+ inhibited by mPT inhibitors such as olesoxime or DUB, showing unpublished results). Besides such lysosomal Ca exit channels, 2+ the role of lysosomal Ca release and consequent mPT in other members of the TRPM family or those belonging to different 2+ lipotoxicity, which is consistent with previous papers reporting families might play a role in Ca -mediated lipotoxic cell death, 2+ mPT induction by PA [2, 38]. Identity of lysosomal Ca exit which could be a subject for further studies. It remains to be Cell Death Discovery (2023) 9:100 S.-J. Oh et al. Fig. 4 SOCE in lipotoxicity. A After treatment of HepG2 cells transfected with YFP-STIM1 and mCherry-Orai1 with PA for 6 h, STIM1 aggregation and its co-localization with ORAI1 were examined at the middle and bottom of the cells by confocal microscopy (left). Scale bar, 10 μm. Pearson’s correlation coefﬁcient was calculated as an index of co-localization of YFP-STIM1 and mCherry-Orai1 (right). B After treatment of 2+ GEM-CEPIA1er-transfected cells with PA in the presence or absence of 10 μM BTP2 for 6 h, [Ca ] was determined by confocal microscopy. ER 2+ C, D After the same treatment as in (B), [Ca ] was determined by confocal microscopy after Fluo-3 AM loading (C) or ratiometric analysis after Fura-2 loading (D). E, F After treatment of cells with PA in the presence or absence of 10 μM BTP2 (E) or 2 mM EGTA (F) for 24 h, cell death was evaluated using SYTOX Green. Data are expressed as the means ± SD of three independent experiments. Statistical comparisons were performed using two-tailed Student’s t-test (A) or one-way ANOVA with Tukey’s multiple comparison test (B–F). (*P < 0.05; **P < 0.01; ***P < 0.001; ns not signiﬁcant). Cell Death Discovery (2023) 9:100 S.-J. Oh et al. Fig. 5 In vivo lipotoxicity. A After injection of ethyl palmitate and LPS to C57BL/6 mice with or without pretreatment with dantrolene or DUB, cell death was evaluated by TUNEL staining of hepatic sections (right). Representative TUNEL staining is presented (left) (n= 8–9/group). Insets were magniﬁed. Scale bar, 200 μm. B In serum from mice of (A), ALT and AST levels were determined using a blood chemistry analyzer (n= 8–9/group). C In frozen hepatic sections from the mice of (A), mitochondrial ROS accumulation was determined by MitoSOX staining (n= 5–6/group) (right). Representative ﬂuorescence images are shown (left). Scale bar, 20 μm. Data are presented as the means ± SD. All statistical comparisons were performed using one-way ANOVA with Tukey’s multiple comparison test. (*P < 0.05; **P <0.01; ***P < 0.001; ns not signiﬁcant). Cell Death Discovery (2023) 9:100 S.-J. Oh et al. 2+ clariﬁed which lysosomal Ca channel is involved in lipotoxicity MATERIALS AND METHODS 2+ due to lysosomal Ca -mediated mPT. Reagents 2+ Reagents in this study were purchased from following sources: Oregon It has been demonstrated that ER→ lysosome Ca ﬂux occurs 2+ 2+ Green-488 BAPTA-1 dextran (OGBD, O6789), Fluo-3 AM (F23915), when lysosomal Ca content is lowered due to lysosomal Ca TM MitoSOX Red (M36008), SYTOX Green (S7020), BAPTA-AM (O6807), release by mitochondrial stressors [12, 31]. Such reﬁlling of lysosomal 2+ 2+ 2+ calcein-AM (C1430), Fura-2 (F1221) from Thermo Fisher Scientiﬁc; palmitate Ca pool from ER Ca pool occurring when lysosomal Ca (PA, P0500), decylubiquinone (DUB, D7911), bovine serum albumin (BSA, content is lowered, is likely due to small lysosomal volume A9418), cobalt chloride (CoCl , 232696), ethylene glycol-bis (2-aminoethy- 2+ 2+ accommodating Ca which is 10% of ER Ca volume [30]. Thus, lether)-N,N,N′,N′-tetraacetic acid (EGTA, 03777), MitoTEMPO (SML0737), CA- 2+ 2+ 2+ while [Ca ] is comparable to [Ca ] , lysosomal Ca content Lys ER 074Me (205531) from Sigma-Aldrich; Gly-Phe β-naphthylamide (GPN, 2+ might not be sufﬁcient for progression of events requiring Ca ﬂux. ab145914) from Abcam; BTP2 (S8380) from Selleckchem; CytoTox 96® 2+ 2+ TM Besides ER Ca pool, another source of lysosomal Ca could be Non-Radioactive Cytotoxicity Assay (G1780) from Promega Corporation. 2+ endocytic pathway. However, most of endocytic Ca has been reported to be dissipated before reaching lysosome [46], supporting Cell culture and treatment 2+ 2+ the importance of ER Ca as a dominant source of lysosomal Ca HepG2 cells obtained from Korean Cell Line Bank (KCLB) were grown in DMEM 2+ pool. The role of ER→ lysosome Ca ﬂux in diverse pathological and medium (Welgene, LM-001-05)-1% penicillin–streptomycin–amphotericin B physiological conditions could be an intriguing topic to be explored mixture (Lonza, 17-745E) supplemented with 10% fetal bovine serum (Corning, 35-010-CV). Cells were tested for mycoplasma contamination using in future studies. When we studied whether similar phenomenon 2+ a Mycoplasma PCR Detection Kit (e-MycoTM, 25236, iNtRON Biotechnology). occurs after PA treatment of HepG2 cells, ER→ lysosome Ca PA stock solution was prepared by dissolving palmitate in 70% ethanol and reﬁlling could be clearly demonstrated by simultaneous monitoring 2+ 2+ 2+ heating at 56 °C. PA stock solution was diluted in 2% fatty acid-free BSA- of [Ca ] and [Ca ] during incubation in a Ca -free medium ER Lys DMEM before treatment. For in vitro treatment, following concentrations were 2+ ensuring recovery of [Ca ] after removal of PA. Furthermore, TM Lys employed: OGBD, 100 μg/ml;Fluo-3AM, 5 μM; MitoSOX Red, 5 μM; SYTOX 2+ blockade of ER→ lysosome Ca reﬁlling with dantrolene, an Green Nucleic Acid Stain, 1 μM; BAPTA-AM, 30 μM; calcein-AM, 1 μM; palmitic 2+ 2+ inhibitor of RyR Ca exit channel on ER or chelation of ER Ca acid (PA), 500 μM; decylubiquinone (DUB), 200 μM; cobalt chloride (CoCl ), by TPEN could inhibit lipotoxicity, which indicates that intracellular 2mM; Gly-Phe β-naphthylamide (GPN), 200 μM; BTP2, 10 μM; MitoTEMPO, 2+ 2+ Ca ﬂux from ER occurs to sustain lysosomal Ca release, resulting 10 μM; EGTA, 2 mM; CA-074Me, 10 μM. 2+ 2+ in cell death. Ca ﬂux from ER to lysosome, in turn, induced ER Ca emptying which activated SOCE. Occurrence of SOCE after PA SYTOX Green nucleic acid staining 2+ TM treatment of HepG2 cells was evidenced by no decrease of [Ca ] ER Cell death was evaluated using SYTOX Green Nucleic Acid Stain kit 2+ in cells treated with PA without removal of extracellular Ca , (Thermo Fisher Scientiﬁc, S7020). Brieﬂy, HepG2 cells were seeded in a 24- 2+ reappearance of the decrease of [Ca ] by BTP2 even in the well plate. After 24 h, cells were treated with PA with or without indicated ER 2+ compound for 24 h. Cells were then incubated with SYTOX Green (1 μM) presence of extracellular Ca and STIM1 aggregation co-localized for 30 min at 37 °C. SYTOX Green ﬂuorescence was measured by ﬂow with ORAI1. Functional role of SOCE after treatment with PA was cytometry using BD FACSVerse and FACSCanto II (BD Biosciences, San Jose, demonstrated by inhibition of lipotoxicity by BTP2 or extracellular 2+ CA, USA). Data analysis was performed using FlowJo software (10.8, FlowJo, Ca chelation with EGTA. Furthermore, occurrence of SOCE and no LLC, BD Biosciences). 2+ decrease of [Ca ] in cells treated with PA without removal of ER 2+ 2+ extracellular Ca supports that increase of [Ca ] after PA treatment 2+ LDH release assay is not due to direct release of Ca from ER through SERCA inhibition, TM 2+ Cell death was assessed using an LDH release assay kit (Promega sinceincrease[Ca ] due to SERCA inhibition would not be affected 2+ Corporation, G1780). Brieﬂy, HepG2 cells seeded 4 × 10 well in a 96-well by extracellular Ca [32]. While SERCA inhibition is an important plate were treated with PA with or without indicated compound for 24 h. component of ER stress associated with lipid overload or obesity Culture supernatant was collected and analyzed according to the in vivo inducing altered membrane phospholipid composition [47], manufacturer’s protocol. SERCA might not be important in acute lipotoxicity of HepG2 cells in vitro. However, we do not eliminate the possibility that ER stress Transfection and plasmids might contribute to lipotoxicity in vitro. In fact, we have observed Cells were transfected with plasmids such as GCaMP3-ML1, GEM-CEPIA1er, that CHOP, an important player in ER stress-induced cell death, can TM YFP-STIM1, 3xFLAG-mCherry Red-Orai1/P3XFLAG7.1 using PolyJet In Vitro be activated by mitochondrial ROS produced after PA treatment [48]. ® DNA Transfection Reagent (SigmaGen Laboratories, SL100688), according 2+ In our experiments to investigate the role of increased Ca and to the manufacturer’s protocol. mPT in lipotoxicity in vivo, we observed that DUB inhibiting mPT 2+ and dantrolene abrogating [Ca ] increase and cell death in vitro 2+ Measurement of cytosolic, ER and lysosome Ca contents by PA could inhibit lipotoxic death of hepatocytes in vivo. 2+ To measure [Ca ] , HepG2 cells grown on a chambered coverglass Lys Reduction of mitochondrial ROS by dantrolene or DUB on (Thermo Fisher Scientiﬁc, 155383) were loaded with 100 μg/ml OGBD, an 2+ mitochondrial ROS could be due to a feed-forward response indicator of lysosomal luminal Ca for 16 h. After incubation in a fresh 2+ leading to further ROS release following Ca -mediated mPT media for 4 h, cells were treated with BSA or 500 μM PA for 6 h. After 2+ [49, 50] which was inhibited by dantrolene or DUB. Dantrolene is a washing with Ca -free HEPES-buffered saline (HBS), ﬂuorescence was measured using an LSM780 confocal microscope (Zeiss, Oberkochen, candidate for therapeutic drug against malignant hyperthermia or Germany) and quantiﬁed using ImageJ software. Alzheimer’s disease [51, 52]. DUB has also been studied as a 2+ To determine [Ca ] by confocal microscopy, HepG2 cells grown on potential therapeutic agent against Friedreich’s ataxia [53]or chambered coverglass were loaded with 5 μM Fluo-3 AM for 30 min, an tumor-induced angiogenesis [54]. Our results suggest the 2+ indicator of cytosolic Ca . After treatment with 500 μM PA for 6 h, cells possibility that dantrolene or DUB could be considered candidates 2+ were washed with Ca -free PBS twice, and ﬂuorescence was recorded for drug agents against diseases characterized by lipotoxicity such using an LSM780 confocal microscope. Fluorescence intensity was 2+ as nonalcoholic steatohepatitis or cardiomyopathy. While we have quantiﬁed using ImageJ software. For ratiometric [Ca ] measurement, 2+ shown the contribution of mPT induced by lysosomal Ca release cells were loaded with 2 μM of the acetoxymethyl ester form of Fura-2 in 2+ as a mechanism of lipotoxicity in vitro and in vivo, other previously DMEM at 37 °C for 30 min and then washed with a basal Ca solution (145 mM NaCl, 5 mM KCl, 3 mM MgCl , 10 mM glucose, 1 mM EGTA, 20 mM reported mechanisms of lipotoxicity such as ER or mitochondrial 2 HEPES, pH 7.4). Measurements were conducted using MetaFluor on an stress and generation of ceramide or lysophosphatidylcholine Axio Observer A1 (Zeiss) equipped with a 150 W xenon lamp Polychrome V [1–4] could also play an important role depending on the cellular (Till Photonics, Bloaaom Drive Victor, NY, USA), a CoolSNAP-Hq2 digital context or environmental condition, and optimal strategy against camera (Photometrics, Tucson, AZ, USA), and a Fura-2 ﬁlter set. lipotoxicity could be different accordingly. Cell Death Discovery (2023) 9:100 S.-J. Oh et al. Fluorescence at 340/380 nm was measured in phenol red-free medium, Humane Care and Use of Laboratory Animals. The protocol was 2+ and converted to [Ca ] using the following equation [23]. approved by the Institutional Animal Care and Use Committee (IACUC) of the Department of Laboratory Animal Resources of Soonchunhyang 2þ Ca ¼ K ´ ½ðÞ R R =ðÞ R R ´ F =F d min max minð380Þ maxð380Þ Institute of Medi-bio Science. Ethyl palmitate (Tokyo Chemical Industry, P0003) was dissolved in water with 4.8% lecithin (FUJIFILM Wako Pure where K = Fura-2 dissociation constant (224 nM at 37 °C), F = the d min(380) Chemical Corporation, 120-00832) and 10% glycerol (Sigma-Aldrich, 2+ 380 nm ﬂuorescence in the absence of Ca , F = 380 nm ﬂuores- max(380) G2025) to make a mixture containing ethyl palmitate at a concentration 2+ cence with saturating Ca , R = 340/380 nm ﬂuorescence ratio, R = max of 300 mM. LPS (Sigma-Aldrich, E. coli O55:B5) was dissolved in PBS, and 2+ 340/380 nm ratio with saturating Ca , and R = 340/380 nm ratio in the min 0.025 mg/kg LPS was injected intraperitoneally into mice. Dantrolene 2+ absence of Ca . andDUB were dissolvedinDMSO anddiluted with PBS. For invivo 2+ 2+ To determine ER Ca contents ([Ca ] ), HepG2 cells were grown on ER administration, dantrolene (10 mg/kg), DUB (5 mg/kg) or DMSO solution chambered coverglass and transfected with a GEM-CEPIA1er plasmid, a diluted in PBS was injected into mice that were fasted for 24 h. After 1 h, 2+ ratiometric ﬂuorescent indicator of ER Ca . After 24 h, cells were 300 mM ethyl palmitate or vehicle was administered, followed by LPS 2+ treated with BSA or 500 μM PA for 6 h, and then washed with Ca -free injection 1 h later. Blood and liver tissue samples were obtained KRBB (Sigma-Aldrich, K4002). GEM-CEPIA1er ﬂuorescence was measured 24 h later. using an LSM780 confocal microscope at an excitation wavelength of 405 nm and an emission wavelength of 466 or 520 nm. Fluorescence 2+ ratio F466/F520 was calculated as an index of [Ca ] [32]. Blood chemistry ER Serum ALT and AST levels were measured using a Fuji Dri-Chem NX500i chemistry Analyzer (Fujiﬁlm, Tokyo, Japan). 2+ GCaMP3-ML1 Ca imaging 2+ To detect perilysosomal Ca release, HepG2 cells were transfected with a 2+ 2+ GCaMP3-ML1 Ca probe, a lysosome-targeted genetically-encoded Ca TUNEL staining sensor. Twenty-four h after transfection, cells were treated with BSA or Parafﬁn-embedded liver tissue blocks were prepared by ﬁxing in 10% 2+ 500 μM PA for 6 h. Perilysosomal Ca release was recorded by monitoring neutral buffered formalin (Sigma, HT501128). TUNEL staining was 2+ GCaMP3 ﬂuorescence intensity at 470 nm in a basal Ca solution (145 mM conducted using In Situ Cell Death Detection kit (Roche, 11684817910) NaCl, 5 mM KCl, 3 mM MgCl , 10 mM glucose, 1 mM EGTA, 20 mM HEPES, according to the manufacturer’s instructions. Cell death index was pH 7.4) using a Zeiss LSM780 confocal microscope. GPN, a lysosomotropic expressed as the number of TUNEL-positive cells per ﬁeld counted from 2+ agent, of 200 μM was added to evoke lysosomal Ca release. GCaMP3- more than 20 ﬁelds randomly selected (×200). ML1 ﬂuorescence was calculated as a change GCaMP3 ﬂuorescence (ΔF) over baseline ﬂuorescence (F ). Statistical analysis Data are presented as means ± SD of three independent experiments. Two- STIM1 and ORAI1 co-localization tailed Student’s t-test was used to compare values between two groups. After transfection with YFP-STIM1 and 3xFLAG-mCherry Red-Orai1/ One-way ANOVA with Tukey’s test was used to compare values between P3XFLAG7.1 (provided by Yuan J through Cha S-G) plasmids for 48 h, multiple groups. Data were considered signiﬁcant when P < 0.05. Data HepG2 cells were treated with BSA or 500 μM PA for 6 h. Cells were then analysis was performed using GraphPad Prism 8 software (GraphPad ﬁxed with 4% paraformaldehyde (PFA) at room temperature for 10 min. Software, La Jolla, CA, USA). Fluorescence images were acquired with an LSM780 confocal microscope, and the co-localization between STIM1 and ORAI1 was examined by calculating Pearson’s correlation coefﬁcient using ZEN software (Carl Zeiss DATA AVAILABILITY Microscopy GmbH, Jena, Germany). The data generated or analyzed during the current study and materials are available from the corresponding author without imposed restriction on reasonable request. Measurement of mitochondrial ROS TM Mitochondrial ROS was measured using MitoSOX Red. Brieﬂy, HepG2 REFERENCES cells were pretreated with 10 μM MitoTEMPO for 1 h, and then treated with TM 500 μM PA for 24 h. After incubation with 5 μM MitoSOX Red at 37 °C for 1. Han MS, Park SY, Shinzawa K, Kim S, Chung KW, Lee JH, et al. Lysopho- 15 min, ﬂuorescence was measured by ﬂow cytometry using BD FACSVerse sphatidylcholine as a death effector in lipoapoptosis of hepatocytes. J Lipid Res. or FACSCanto II. Data were analyzed using FlowJo software. 2008;49:84–97. To determine mitochondrial ROS accumulation in vivo, frozen sections 2. Koshkin V, Dai FF, Robson-Doucette CA, Chan CB, Wheeler MB. Limited mito- TM of the liver tissue were incubated with MitoSOX Red at 37 °C for 15 min, chondrial permeabilization is an early manifestation of palmitate-induced lipo- and then washed with PBS. Fluorescence images were observed with an toxicity in pancreatic beta-cells. J Biol Chem. 2008;283:7936–48. LSM710 confocal microscope and ﬂuorescence intensity was quantiﬁed 3. Shimabukuro M, Wang MY, Zhou YT, Newgard CB, Unger RH. Protection against using ImageJ software (NIH, Bethesda, MD, USA). lipoapoptosis of b cells through leptin-dependent maintenance of Bcl-2 expression. Proc Natl Acad Sci USA. 1998;95:9558–61. 4. Sommerweiss D, Gorski T, Richter S, Garten A, Kiess W. Oleate rescues INS-1E mPTP opening 2+ β-cells from palmitate-induced apoptosis by preventing activation of the unfol- mPTP opening was assessed by Co quenching of calcein-AM ﬂuores- ded protein response. Biochem Biophys Res Com. 2013;441:770–6. cence. Cells were treated with PA (500 μM) with or without DUB (200 μM) 5. Katsoulieris E, Mabley JG, Samai M, Green IC, Chatterjee PK. alpha-Linolenic acid or olesoxime (100 μM) for 24 h. Cells were then loaded with calcein-AM protects renal cells against palmitic acid lipotoxicity via inhibition of endoplasmic (1 μM) and CoCl (2 mM) at 37 °C for 15 min. Calcein ﬂuorescence was reticulum stress. Eur J Pharmacol. 2009;623:108–12. measured by ﬂow cytometry employing BD FACSVerse and FACSCanto II 6. Khan MJ, Alam MR, Waldeck-Weiermair M, Karsten F, Groschner L, Riederer M, and analyzed using FlowJo software. et al. Inhibition of autophagy rescues palmitic acid-induced necroptosis of endothelial cells. J Biol Chem. 2012;287:21110–20. Mitochondrial membrane potential 7. Shimabukuro M, Zhou YT, Levi M, Unger RH. Fatty acid-induced b cell apoptosis: a Mitochondrial membrane potential was determined using tetramethylrhoda- link between obesity and diabetes. Proc Natl Acad Sci USA. 1998;95:2498–502. mine ethyl ester perchlorate (TMRE). After treatment of HepG2 cells with PA 8. Sparagna GC, Hickson-Bick DL, Buja LM, McMillin JB. A metabolic role for mito- for 24 h, cells were incubated with 100 nM TMRE at 37 °C for 30 min, and chondria in palmitate-induced cardiac myocyte apoptosis. Am J Physiol. ﬂuorescence was measured using LSM710 confocal microscope. Fluorescence 2000;279:H2124–2132. intensity was quantiﬁed using ImageJ software. 9. Xu S, Nam SM, Kim JH, Das R, Choi SK, Nguyen TT, et al. Palmitate induces ER calcium depletion and apoptosis in mouse podocytes subsequent to mito- chondrial oxidative stress. Cell Death Dis. 2015;6:e1976. Animals 10. Karasawa T, Kawashima A, Usui-Kawanishi F, Watanabe S, Kimura H, Kamata R, Eight-week-old C57BL/6N male mice were purchased from Orient Bio et al. Saturated fatty acids undergo intracellular crystallization and activate the (Seongnam, Korea). All experiments using mice were performed in NLRP3 inﬂammasome in macrophages. Arterioscler Thromb Vasc Biol. accordance with the guidelines of the Public Health Service Policy in 2018;38:744–56. Cell Death Discovery (2023) 9:100 S.-J. Oh et al. 11. Kim J, Kim SH, Kang H, Lee S, Park S-Y, Cho Y, et al. TFEB-GDF15 axis protects 40. Du W, Gu M, Hu M, Pinchi P, Chen W, Ryan M, et al. Lysosomal Zn 2+ release against obesity and insulin resistance as a lysosomal stress response. Nature triggers rapid, mitochondria-mediated, non-apoptotic cell death in metastatic Metabolism. 2021;3:410–27. melanoma. Cell Rep. 2021;37:109848. 12. Park K, Lim H, Kim J, Hwang H, Lee YS, Bae SH, et al. Essential role of lysosomal 41. Zaibi N, Li P, Xu S-Z. Protective effects of dapagliﬂozin against oxidative stress- Ca2+-mediated TFEB activation in mitophagy and functional adaptation of induced cell injury in human proximal tubular cells. PLoS ONE. 2021;16:e0247234. pancreatic β-cells to metabolic stress. Nat Commun. 2022;13:1300. 42. Kraft R, Grimm C, Frenzel H, Harteneck C. Inhibition of TRPM2 cation channels by 13. Trudeau KM, Colby AH, Zeng J, Las G, Feng JH, Grinstaff MW, et al. Lysosome N-(p-amylcinnamoyl)anthranilic acid. Br J Pharma. 2006;148:264–73. acidiﬁcation by photoactivated nanoparticles restores autophagy under lipo- 43. Samie M, Wang X, Zhang X, Goschka A, Li X, Cheng X, et al. A TRP channel in the toxicity. J Cell Biol. 2016;214:25–34. lysosome regulates large particle phagocytosis via focal exocytosis. Dev Cell. 14. Fontaine E, Ichas F, Bernardi P. A ubiquinone-binding site regulates the mito- 2013;26:511–24. chondrial permeability transition pore. J Biol Chem. 1998;273:25734–40. 44. Sun J, Liu Y, Hao X, Lin W, Su W, Chiang E, et al. LAMTOR1 inhibition of TRPML1- 15. Nakagawa T, Shimizu S, Watanabe T, Yamaguchi O, Otsu K, Yamagata H, et al. dependent lysosomal calcium release regulates dendritic lysosome trafﬁcking Cyclophilin D-dependent mitochondrial permeability transition regulates some and hippocampal neuronal function. EMBO J. 2022;41:e108119. necrotic but not apoptotic cell death. Nature. 2005;434:652–8. 45. Simon JS, Vrellaku B, Monterisi S, Chu SM, Rawlings N, Lomas O, et al. Oxidation of 16. Schönfeld P, Wojtczak L. Fatty acids as modulators of the cellular production of protein kinase A regulatory subunit PKARIα porotects against myocardial reactive oxygen species. Free Radic Biol Med. 2007;45:231–41. ischemia-reperfusion injury byinhibiting lysosomal-triggered calcium release. 17. Zhang X, Cheng X, Yu L, Yang J, Calvo R, Patnaik S, et al. MCOLN1 is a ROS sensor Circulation. 2020;143:449–65. in lysosomes that regulates autophagy. Nat Commun. 2016;7:12109. 46. Gerasimenko JV, Tepikin AV, Petersen OH, Gerasimenko OV. Calcium uptake via 18. Chang I, Cho N, Kim S, Kim JY, Kim E, Woo JE, et al. Role of calcium in pancreatic endocytosis with rapid release from acidifying endosomes. Curr Biol. 1998;8:1335–8. islet cell death by IFN-gamma/TNF-alpha. J Immunol. 2004;172:7008–14. 47. Fu S, Yang L, Li P, Hofmann O, Dicker L, Hide W, et al. Aberrant lipid metabolism 19. Maher P, van Leyen K, Dey PN, Honrath B, Dolga A, Methner A. The role of Ca 2+ disrupts calcium homeostasis causing liver endoplasmic reticulum stress in in cell death caused by oxidative glutamate toxicity and ferroptosis. Cell Calcium. obesity. Nature. 2011;473:528–31. 2018;70:47–55. 48. Ly LD, Ly DD, Nguyen NT, Kim J-H, Yoo H, Chung J, et al. Mitochondrial Ca2+ 20. Zhivotovsky B, Orrenius S. Calcium and cell death mechanisms: a perspective uptake relieves palmitate-induced cytosolic Ca2+ overload in MIN6 cells. Mol from the cell death community. Cell Calcium. 2011;50:211–21. Cells. 2020;43:66–75. 21. Hunnicutt JH, Hardy RW, Williford J, McDonald JM. Saturated fatty acid-induced 49. Zorov DB, Filburn CR, Klotz LO, Zweier JL, Sollott SJ. Reactive oxygen species insulin resistance in rat adipocytes. Diabetes. 1994;43:540–5. (ROS)-induced ROS release: a new phenomenon accompanying induction of the 22. Shen D, Wang X, Li X, Zhang X, Yao Z, Dibble S, et al. Lipid storage disorders block mitochondrial permeability transition in cardiac myocytes. J Exp Med. lysosomal trafﬁcking by inhibiting a TRP channel and lysosomal calcium release. 2000;192:1001–14. Nat Commun. 2012;3:731. 50. Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial reactive oxygen species (ROS) 23. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with and ROS-induced ROS release. Physiol Rev. 2014;94:909–50. greatly improved ﬂuorescence properties. J Biol Chem. 1985;260:3440–50. 51. Karagas NE, Venkatachalam K. Roles for the endoplasmic reticulum in regulation 24. Li Z, Berk M, McIntyre TM, Gores GJ, Feldstein AE. The lysosomal-mitochondrial of neuronal calcium homeostasis. Cells. 2019;8:1232. axis in free fatty acid-induced hepatic lipotoxicity. Hepatology. 2008;47:1495–503. 52. Kim KSM, Kriss RS, Tautz TJ. Malignant hyperthermia: a clinical review. Adv 25. Rial E, Rodríguez-Sánchez L, Gallardo-Vara E, Zaragoza P, Moyano E, González- Anesth. 2019;37:35–51. Barroso MM. Lipotoxicity, fatty acid uncoupling and mitochondrial carrier func- 53. Jauslin ML, Meier T, Smith RA, Murphy MP. Mitochondria-targeted antioxidants tion. Biochim Biophys Acta. 2008;1979:800–6. protect Friedreich Ataxia ﬁbroblasts from endogenous oxidative stress more 26. Petronilli V, Miotto G, Canton M, Colonna R, Bernardi P, Di Lisa F. Imaging the effectively than untargeted antioxidants. FASEB J. 2003;17:1972–4. mitochondrial permeability transition pore in intact cells. Biofactors. 1998;8:263–72. 54. Cao J, Liu X, Yang Y, Wei B, Li Q, Mao G, et al. Decylubiquinone suppresses breast 27. Bradham CA, Qian T, Streetz K, Trautwein C, Brenner DA, Lemasters JJ. The cancer growth and metastasis by inhibiting angiogenesis via the ROS/p53/ mitochondrial permeability transition is required for tumor necrosis factor alpha- BAI1 signaling pathway. Angiogenesis. 2020;23:325–38. mediated apoptosis and cytochrome c release. Mol Cell Biol. 1998;18:6353–64. 28. Halestrap AP, Kerr PM, Javadov S, Woodﬁeld KY. Elucidating the molecular mechanism of the permeability transition pore and its role in reperfusion injury of the heart. Biochim Biophys Acta. 1998;1366:79–94. AUTHOR CONTRIBUTIONS 29. Bordet T, Berna P, Abitbol J-L, Pruss RM. Olesoxime (TRO19622): a novel M-SL and KYH conceived the experiments. S-JO and YH conducted the experiments. mitochondrial-targeted neuroprotective compound. Pharmaceuticals. 2010;3:345–68. M-SL, KYH, S-JO and YH wrote the manuscript. 30. Penny CJ, Kilpatrick BS, Han JM, Sneyd J, Patel S. A computational model of lysosome-ER Ca2+ microdomains. J Cell Sci. 2014;127:2934–43. 31. Garrity AG, Wang W, COllier CMD, Levery SA, Gao Q, Xu H. The endoplasmic reticulum, not the pH gradient, drives calcium reﬁlling of lysosomes. eLife. FUNDING 2016;5:e15887. This study was supported by a National Research Foundation of Korea (NRF) grant 32. Suzuki J, Kanemaru K, Ishii K, Ohkura M, Lino M. Imaging intraorganellar Ca2+ at funded by the Korea government (MSIT) (NRF-2019R1A2C3002924) and by the subcellular resolution using CEPIA. Nat Commun. 2014;5:4153. Bio&Medical Technology Development Program (2017M3A9G7073521). M-S Lee is 33. Derler I, Jardin I, Romanin C. Molecular mechanisms of STIM/Orai communication. the recipient of Korea Drug Development Fund from the Ministry of Science & ICT, Am J Physiol. 2016;310:C643–662. Ministry of Trade, Industry & Energy and Ministry of Health & Welfare (HN22C0278), 34. Hofer AM, Fasolato C, Pozzan T. Capacitative Ca2+ entry is closely linked to the and a grant from the Faculty Research Assistance Program of Soonchunhyang ﬁlling state of internal Ca2+ stores: a study using simultaneous measurements of University (20220346). ICRAC and Intraluminal [Ca2+]. J Cell Biol. 1998;130:325–34. 35. Bogeski I, Al-Ansary D, Qu B, Niemeyer BA, Hoth M, Peinelt C. Pharmacology of ORAI channels as a tool to understand their physiological functions. Expert Rev Clin Pharmacol. 2010;3:291–303. COMPETING INTERESTS 36. Zitt C, Strauss B, Schwarz EC, Spaeth N, Rast G, Hatzelmann A, et al. Potent M-SL in the CEO of LysoTech, Inc. The authors declare no competing interests. inhibition of Ca2+ release-activated Ca2+ channels and T-lymphocyte activation by the pyrazole derivative BTP2. J Biol Chem. 2004;279:12427–37. 37. Ogawa Y, Imajo K, Honda Y, Kessoku T, Tomeno W, Kato S, et al. Palmitate- induced lipotoxicity is crucial for the pathogenesis of nonalcoholic fatty liver ADDITIONAL INFORMATION disease in cooperation with gut-derived endotoxin. Sci Rep. 2018;8:11365. Correspondence and requests for materials should be addressed to Myung-Shik Lee. 38. Srivastava S, Chan C. Hydrogen peroxide and hydroxyl radicals mediate palmitate-induced cytotoxicity to hepatoma cells: relation to mitochondrial Reprints and permission information is available at http://www.nature.com/ permeability transition. Free Radic Res. 2007;41:38–49. reprints 39. Akyuva Y, Nazıroğlu M. Resveratrol attenuates hypoxia-induced neuronal cell death, inﬂammation and mitochondrial oxidative stress by modulation of TRPM2 Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims channel. Sci Rep. 2020;10:6449. in published maps and institutional afﬁliations. Cell Death Discovery (2023) 9:100 S.-J. Oh et al. 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Copyright: Copyright © The Author(s) 2023
eISSN: 2058-7716
DOI: 10.1038/s41420-023-01379-0
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Abstract

www.nature.com/cddiscovery ARTICLE OPEN 2+ Lysosomal Ca as a mediator of palmitate-induced lipotoxicity 1,2,5 3,5 4 2,3 Soo-Jin Oh , Yeseong Hwang , Kyu Yeon Hur and Myung-Shik Lee © The Author(s) 2023 While the mechanism of lipotoxicity by palmitic acid (PA), an effector of metabolic stress in vitro and in vivo, has been extensively investigated, molecular details of lipotoxicity are still not fully characterized. Since recent studies reported that PA can exert lysosomal stress in addition to well-known ER and mitochondrial stress, we studied the role of lysosomal events in lipotoxicity by 2+ PA, focusing on lysosomal Ca . We found that PA induced accumulation of mitochondrial ROS and that mitochondrial ROS 2+ 2+ 2+ induced release of lysosomal Ca due to lysosomal Ca exit channel activation. Lysosomal Ca release led to increased cytosolic 2+ 2+ Ca which induced mitochondrial permeability transition (mPT). Chelation of cytoplasmic Ca or blockade of mPT with olesoxime 2+ 2+ or decylubiquinone (DUB) suppressed lipotoxicity. Lysosomal Ca release led to reduced lysosomal Ca content which was 2+ 2+ 2+ replenished by ER Ca , the largest intracellular Ca reservoir (ER→ lysosome Ca reﬁlling), which in turn activated store- 2+ 2+ 2+ operated Ca entry (SOCE). Inhibition of ER→ lysosome Ca reﬁlling by blockade of ER Ca exit channel using dantrolene or inhibition of SOCE using BTP2 inhibited lipotoxicity in vitro. Dantrolene or DUB also inhibited lipotoxic death of hepatocytes in vivo induced by administration of ethyl palmitate together with LPS. These results suggest a novel pathway of lipotoxicity characterized 2+ 2+ by mPT due to lysosomal Ca release which was supplemented by ER→ lysosome Ca reﬁlling and subsequent SOCE, and also 2+ 2+ suggest the potential role of modulation of ER→ lysosome Ca reﬁlling by dantrolene or other blockers of ER Ca exit channels in disease conditions characterized by lipotoxicity such as metabolic syndrome, diabetes, cardiomyopathy or nonalcoholic steatohepatitis. Cell Death Discovery (2023) 9:100 ; https://doi.org/10.1038/s41420-023-01379-0 INTRODUCTION RESULTS 2+ Palmitic acid (PA) is an important molecule acting as an effector PA elicits lysosomal Ca release of lipid injury and metabolic stress in vitro and in vivo. PA can induce reactive oxygen species (ROS) by compromising Mechanisms of PA-induced lipotoxicity have been ascribed to mitochondrial complex I and III [11, 16], which can activate lysosomal 2+ 2+ 2+ endoplasmic reticulum (ER) stress, mitochondrial stress, pro- Ca channel [12, 17]. Lysosomal Ca release due to lysosomal Ca 2+ 2+ duction of ceramide or lysophosphatidylcholine [1–4], leading exit channel activation can increase cytosolic Ca content ([Ca ] ), to JNK activation, apoptosis or necrosis, depending on the which, in turn, can induce mitophagy, lysosomal stress response experimental condition and cellular context [3, 5–7]. While ER [11, 12] or diverse types of cell death [18–20]. Hence, we studied and mitochondria are well-established target organelles of PA- whether PA-induced lipotoxicity entails mitochondrial ROS-induced 2+ 2+ induced injury [2, 4, 8, 9], lysosomal changes have recently been lysosomal Ca release and increased [Ca ] . reported to occur such as decreased lysosomal acidity, altered When we treated HepG2 cells with PA, signiﬁcant lipotoxic cell lysosomal enzyme activity or impaired lysosomal integrity death was observed in a dose range of 500 ~ 1,000 μM, as 2+ [10–13]. We recently reported that lysosomal Ca is released revealed by SYTOX Green staining (Fig. 1A). Furthermore, after treatment with PA, while the cellular consequences of accumulation of mitochondrial ROS stained with MitoSOX was 2+ lysosomal Ca releasecouldbedistinctdepending on thecell well visualized in the same dose range of PA, which was types or context of treatment [11, 12]. Since release of signiﬁcantly reduced by MitoTEMPO, a mitochondrial ROS 2+ 2+ lysosomal Ca can also lead to increased cytosolic Ca quencher (Fig. 1B), indicating mitochondrial ROS generation by 2+ 2+ content ([Ca ] )[11, 12]and Ca is one of the most important PA. As PA doses higher than 500 μM could be unphysiologically inducers of mitochondrial permeability transition (mPT) and high [21], we employed 500 μM of PA in the following subsequent cell death [14, 15], we conducted this investigation experiments to minimize unphysiological effects of high dose of 2+ based on our hypothesis that lysosomal Ca release by PA can PA. In an experiment to ﬁnd causal relationship between lipotoxic lead to lipotoxic cell death through mPT. cell death and mitochondrial ROS, PA-induced death of HepG2 1 2 Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul 06355, Korea. Department of Integrated Biomedical Science, Soonchunhyang Institute of Medi-bio Science and Division of Endocrinology, Department of Internal Medicine, Soonchunhyang Medical Center, Soonchunhyang University College of Medicine, Cheonan, Korea. Severance Biomedical Science Institute, Graduate school of Medical Science, BK21 Project, Yonsei University College of Medicine, Seoul 03722, Korea. 4 5 Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. These authors contributed equally: Soo-Jin Oh, Yeseong Hwang. email: mslee0923@sch.ac.kr Edited by Professor Myung-Shik Lee Received: 4 September 2022 Revised: 22 February 2023 Accepted: 23 February 2023 Ofﬁcial journal of CDDpress 1234567890();,: S.-J. Oh et al. cells was signiﬁcantly reduced by MitoTEMPO (Fig. 1C), suggesting stained with Oregon Green-488 BAPTA-1 dextran (OGBD) was that mitochondrial ROS generation is a critical event in PA-induced signiﬁcantly decreased in HepG2 cells treated with PA (Fig. 1D), 2+ 2+ lipotoxicity. When we studied possible changes of lysosomal Ca suggesting release of lysosomal Ca by PA treatment, similar to 2+ 2+ after PA treatment, concentration of lysosomal Ca ([Ca ] ) the results using other types of cells [11, 12]. When we indirectly Lys Cell Death Discovery (2023) 9:100 S.-J. Oh et al. 2+ Fig. 1 PA reduces lysosomal Ca .A After treatment of HepG2 cells with 250–1000 μM PA for 24 h, cell death was determined using SYTOX Green. B After treatment of HepG2 cells with 250–1000 μM PA for 24 h in the presence or absence of 10 μM MitoTEMPO, PA-induced mitochondrial ROS was measured by MitoSOX followed by ﬂow cytometry (lower panel). Representative scattergrams are shown (upper panel). C After treatment of HepG2 cells with 500 μM PA for 24 h in the presence or absence of 10 μM MitoTEMPO, cell death was determined using SYTOX Green. D Cells were loaded with Oregon Green-488 BAPTA-1 Dextran (OGBD) for 16 h and chased for 4 h. Cells were then treated 2+ with PA or BSA for 6 h in the presence or absence of 10 μM MitoTEMPO, followed by confocal microscopy to determine [Ca ] (right). Lys Representative ﬂuorescence images are shown (left panel). Scale bar, 20 μm. E After staining with Fluo-3 AM for 30 min, cells were treated in 2+ 2+ PA or BSA for 6 h, followed by confocal microscopy to monitor GPN-induced changes of lysosomal Ca release and [Ca ] increase (left). AUC 2+ of the curve was calculated as an estimate of lysosomal Ca content (right). F Cells transfected with GCaMP3-ML1 were treated with PA or BSA 2+ for 6 h, and then GPN-induced change of GCaMP3-ML1 ﬂuorescence was determined by confocal microscopy to visualize perilysosomal Ca 2+ release (right). Representative ﬂuorescence images are shown (left panel). Scale bar, 10 μm. G [Ca ] in HepG2 cells treated with PA for 6 h was determined by staining with Fluo-3 AM (middle) or ratiometric analysis after Fura-2 loading (right). Representative Fluo-3 ﬂuorescence images 2+ are shown (left panel). Scale bar, 20 μm. H [Ca ] in HepG2 cells treated with PA for 6 h in the presence or absence of 10 μM MitoTEMPO was determined by ratiometric analysis after Fura-2 loading. I After treatment of HepG2 cells with PA in the presence or absence of 10 μM CA- 074Me for 24 h, cell death was determined using SYTOX Green. Data are expressed as the means ± SD of three independent experiments. Statistical comparisons were performed using one-way ANOVA with Tukey’s multiple comparison test (A–D, H, I) or two-tailed unpaired Student’s t-test (vehicle and PA comparisons in E–G). (*P < 0.05; **P < 0.01; ***P < 0.001; ns not signiﬁcant). 2+ estimated lysosomal Ca content by calculating area under the can lead to cell death such as necrosis or apoptosis depending on 2+ 2+ curve (AUC) of cytosolic Ca concentration ([Ca ] ) tracing using the severity of mPT and cellular context [15, 27, 28], we studied Fluo-3 AM staining after treatment with Gly-Phe β-naphthylamide the role of mPT in HepG2 cell death by PA using inhibitors of mPT. 2+ (GPN), a lysosomotropic agent [22], lysosomal Ca content was Indeed, mPT inhibitors such as olesoxime [29] or decylubiquinone again signiﬁcantly reduced by PA treatment (Fig. 1E). Decrease of (DUB) [14], signiﬁcantly reduced PA-induced cell death assessed 2+ [Ca ] was abrogated by MitoTEMPO (Fig. 1D), suggesting that by SYTOX Green staining or LDH release assay (Fig. 2B, C). Reduced Lys mitochondrial ROS produced by PA treatment induces lysosomal lipotoxic cell death by olesoxime or DUB was accompanied by 2+ 2+ 2+ Ca release. We also studied whether perilysosomal Ca release signiﬁcantly reduced mPT as evidenced by decreased Co can be observed after PA treatment employing GCaMP3-ML1, a quenching of calcein ﬂuorescence (Fig. 2D, E), indicating that 2+ 2+ probe detecting perilysosomal Ca release [22]. In GCaMP3-ML1- Ca -mediated mPTP opening is an important mechanism of PA- 2+ transfected HepG2 cells, perilysosomal Ca release was not induced lipotoxicity. PA-induced HepG2 cell death was also directly visualized by PA treatment (Fig. 1F). However, perilysoso- signiﬁcantly reduced by BAPTA-AM, conﬁrming the role of 2+ 2+ 2+ mal Ca release after treatment with GPN was markedly reduced increased [Ca ] and Ca -mediated mPTP in lipotoxicity of 2+ (Fig. 1F), suggesting pre-emptying or release of lysosomal Ca HepG2 cells (Fig. 2F). As a consequence of mitochondrial damage after PA treatment, similar to the results using other types of cells associated with mPT, mitochondrial potential determined by [12, 17]. TMRE staining was signiﬁcantly lowered by PA treatment of 2+ When we determined [Ca ] and studied whether release of HepG2 cells (Fig. 2G). 2+ 2+ 2+ lysosomal Ca leads to increased cytosolic Ca content ([Ca ] ), 2+ [Ca ] estimated by Fluo-3 AM staining was signiﬁcantly ER→ lysosome calcium ﬂux and SOCE in lipotoxicity 2+ 2+ increased by PA (Fig. 1G), supporting release of lysosomal Ca While we showed the role of lysosomal Ca release in lipotoxicity, 2+ 2+ 2+ to the cytosol. When ratiometric measurement of [Ca ] after lysosomal Ca might not be a sufﬁcient source of Ca required 2+ Fura-2 staining was conducted to avoid interference due to for full execution of cellular process requiring Ca , since 2+ 2+ uneven loading or photobleaching [23], increased [Ca ] by PA lysosomal volume is 1% of cell volume and lysosomal Ca pool 2+ treatment was again well observed, which was reversed by is a relatively small Ca reservoir [30]. We thus wondered 2+ MitoTEMPO (Fig. 1G, H), indicating that mitochondrial ROS- whether release or emptying of lysosomal Ca content in HepG2 2+ 2+ induced lysosomal Ca release leads to increased [Ca ] . Since cells treated with PA could be replenished from endoplasmic 2+ 2+ molecules other than Ca might be released from lysosome and reticulum (ER), the largest intracellular Ca reservoir [31] which 2+ could affect cell viability, we studied effect of an inhibitor of has been observed when lysosomal Ca emptying occurs (i.e., 2+ 2+ cathepsin B that has been reported to be released from ER→ lysosome Ca reﬁlling) [12, 31]. When [Ca ] was ER hepatocytes after PA treatment and to induce lipotoxic cell death determined after PA treatment of HepG2 cells using GEM- 2+ [24]. Ca-074Me, a cell-permeable inhibitor of cathepsin B, did not CEPIA1er [32]in a Ca -free Krebs-Ringer bicarbonate (KRB) buffer 2+ 2+ inhibit cell death by PA, suggesting no role of lysosomal cathepsin to abolish possible store-operated Ca entry (SOCE) [33], Ca 2+ B release in lipotoxic cell death of HepG2 cells (Fig. 1I). Taken concentration in the ER ([Ca ] ) was signiﬁcantly reduced (Fig. ER 2+ together, these data demonstrate that PA-induced mitochondrial 3A), suggesting that ER Ca is mobilized probably to replenish 2+ 2+ 2+ ROS leads to release of lysosomal Ca to cytoplasm and lysosomal Ca loss. When [Ca ] was determined without ER 2+ 2+ 2+ increased [Ca ] , contributing to lipotoxic HepG2 cell death. removal of extracellular Ca , [Ca ] was not signiﬁcantly i ER 2+ reduced likely due to activation of store-operated Ca entry 2+ 2+ mPT pore (mPTP) opening mediates lysosomal Ca loss- (SOCE) after ER Ca emptying (Fig. 3A), which suggests that driven cell death in response to PA SERCA was not inhibited by PA since SERCA inhibition reduces 2+ 2+ 2+ We next studied whether PA-induced release of Ca from [Ca ] regardless of extracellular Ca [32] and eliminates the ER 2+ 2+ lysosome into cytosol can induce mPTP opening because Ca is possibility of increased [Ca ] after PA treatment due to SERCA one of the most important inducers of mPT which can lead to inhibition. 2+ mitochondrial catastrophe and cell death [2, 25]. When cells were Since these results suggested replenishment of lysosomal Ca 2+ stained by calcein-AM/CoCl staining, mPTP opening visualized by depletion by ER Ca , we next studied whether ER to lysosomal 2+ 2+ Co quenching of mitochondrial matrix calcein ﬂuorescence [26] Ca movement indeed occurs after PA treatment inducing 2+ was well detected after PA treatment of HepG2 cells, showing lysosomal Ca release. When PA was removed after treatment for 2+ occurrence of mPT by PA (Fig. 2A). PA-induced mPT was 6 h, a decrease of [Ca ] by PA treatment was recovered (Fig. Lys 2+ 2+ abrogated by BAPTA-AM chelating intracellular Ca (Fig. 2A), 3B). To study the role of ER Ca in the recovery of reduced 2+ 2+ 2+ indicating Ca -dependent mPT after PA treatment. Since mPT [Ca ] , we studied the effect of blockade of ER Ca exit Lys Cell Death Discovery (2023) 9:100 S.-J. Oh et al. 2+ channels that could be routes of ER to lysosomal Ca movement hand, dantrolene, an antagonist of ryanodine receptor (RyR), 2+ 2+ during the recovery of [Ca ] . When we employed Xestospon- another ER Ca exit channel, signiﬁcantly suppressed the Lys 2+ 2+ gin C, an IP3R antagonist, recovery of decreased [Ca ] after recovery of suppressed [Ca ] after removal of PA (Fig. 3B), Lys Lys 2+ removal of PA was not signiﬁcantly affected (Fig. 3B). On the other suggesting involvement of RyR channel in ER→ lysosome Ca Cell Death Discovery (2023) 9:100 S.-J. Oh et al. 2+ Fig. 2 mPTP opening by PA due to lysosomal Ca release. A After treatment of HepG2 cells with 500 μM PA in the presence or absence of 2+ 30 μM BAPTA-AM for 24 h, mPTP opening was assessed by ﬂow cytometry after calcein-AM loading and Co quenching (right). Representative histograms are shown (left). B, C After treatment of HepG2 cells with PA in the presence or absence of 100 μM olesoxime (B)or 200 μM DUB (C) for 24 h, cell death was determined using SYTOX Green (left) or LDH release assay (right). D, E After treatment of HepG2 cells with PA in the presence or absence of 100 μM olesoxime (D) or 200 μM DUB (E) for 24 h, mPTP opening was determined as in (A) (right). Representative histograms are shown (left). F After the same treatment as in (A), cell death was determined using SYTOX Green (left) or LDH release assay (right). G After treatment of HepG2 cells with PA as in (A), mitochondrial potential was determined by TMRE staining (right). Representative ﬂuorescence images are shown (left). Scale bar, 20 μm. Data are expressed as the means ± SD of three independent experiments. All statistical comparisons were performed using one-way ANOVA with Tukey’s multiple comparison test. (*P < 0.05; **P < 0.01; ***P < 0.001; ns not signiﬁcant). 2+ reﬁlling after lysosome Ca release by PA. When we chelated ER by PA determined by Fluo-3 AM staining or ratiometric measure- 2+ Ca with a membrane-permeant metal chelator N,N,N’,N’-tetrakis ment after Fura-2 loading was signiﬁcantly attenuated by BTP2 2+ 2+ (2-pyridylmethyl) ethylene diamine (TPEN) that has a low Ca (Fig. 4C, D), indicating the role of SOCE in the increase of [Ca ] by 2+ 2+ afﬁnity and can chelate ER Ca but not cytosolic Ca [34], PA. Furthermore, PA-induced death of HepG2 cells was signiﬁ- 2+ recovery of [Ca ] after removal of PA was signiﬁcantly cantly reduced by BTP2 (Fig. 4E), supporting the role of SOCE in Lys 2+ 2+ 2+ inhibited (Fig. 3B), again supporting the role of ER Ca in the Ca -mediated lipotoxicity. EGTA chelating extracellular Ca also 2+ 2+ recovery of lysosomal Ca . signiﬁcantly reduced increase of [Ca ] and death of HepG2 cells 2+ We next studied the effect of blockade of ER Ca exit channels after PA treatment (Fig. 4C, F), further supporting the role of 2+ 2+ 2+ on the increase of [Ca ] after PA treatment. Again, dantrolene extracellular Ca ﬂux in the increase of [Ca ] and subsequent i i but not Xestospongin C, signiﬁcantly suppressed the increase of lipotoxicity. 2+ [Ca ] after PA treatment determined by ratiometric measure- ment following Fura-2 loading (Fig. 3C), suggesting that ER→ In vivo lipotoxicity 2+ 2+ lysosome Ca reﬁlling through RyR channel contributes to the Based on our in vitro results showing the role of lysosomal Ca 2+ 2+ increase of [Ca ] after PA treatment by replenishing decreased release coupled with ER→ lysosome Ca reﬁlling in mPT and 2+ 2+ lysosomal Ca content and sustaining lysosomal Ca release. cell death in vitro, we next studied the effect of blockade of 2+ 2+ TPEN that reduced recovery of decreased [Ca ] also attenuated lysosomal Ca -mediated mPT on lipotoxicity in vivo. When Lys 2+ increase of [Ca ] after PA treatment (Fig. 3C). When cell death mice were injected with ethyl palmitate followed by LPS was determined, dantrolene but not Xestospongin C, signiﬁcantly administration, death of hepatocytes was observed by TUNEL suppressed the cell death after PA treatment (Fig. 3D), suggesting staining which was accompanied by elevated serum alanine 2+ that ER→ lysosome Ca reﬁlling through RyR channel contributes aminotransferase (ALT)/aspartate aminotransferase (AST) (Fig. to the PA-induced cell death probably by supporting continuous 5A, B), similar to a previous paper [37]. When dantrolene that 2+ increase of cytosolic Ca and subsequent mPTP opening. TPEN was able to inhibit PA-induced hepatocyte death through 2+ also alleviated HepG2 cell death by PA (Fig. 3D), substantiating blockade of the increase of [Ca ] was administered to mice 2+ supportive role of ER Ca in lipotoxicity. To study dynamic before ethyl palmitate injection, death of hepatocytes identiﬁed 2+ changes of [Ca ] and its temporal relationship with lysosomal by TUNEL staining was signiﬁcantly ameliorated, suggesting ER 2+ 2+ 2+ Ca reﬁlling, we simultaneously traced [Ca ] and [Ca ] in blockade of in vivo lipotoxicity by dantrolene (Fig. 5A). Elevated ER Lys cells transfected with GEM-CEPIA1er and loaded with OGBD. When serum ALT/AST levels after ethyl palmitate followed by LPS 2+ organelle [Ca ] was monitored in cells that have reduced administration were also signiﬁcantly reduced by dantrolene 2+ [Ca ] after PA treatment for 6 h and subsequently were pretreatment (Fig. 5B). In addition, accumulation of mitochon- Lys 2+ 2+ incubated in a Ca -free medium, a decrease of [Ca ] occurred drial ROS stained by MitoSOX was observed in the liver tissue of ER 2+ in parallel with an increase of [Ca ] (Fig. 3E), which strongly mice treated with LPS + ethyl palmitate (Fig. 5C), which is in line Lys 2+ supports that ER→ lysosome Ca reﬁlling occurs during HepG2 with mitochondrial ROS accumulation in HepG2 cells treated cell lipotoxicity. with PA in vitro. Such mitochondrial ROS accumulation in vivo 2+ Decrease of [Ca ] after PA treatment only in the absence of was ameliorated by dantrolene administration (Fig. 5C). We also ER 2+ extracellular Ca suggested SOCE without SERCA inhibition studied whether blockade of mPT could inhibit lipotoxicity 2+ because SERCA inhibition would decrease [Ca ] regardless of in vivo based on our in vitro results showing the role of mPT in ER 2+ extracellular Ca [32]. Thus, we next studied possible occurrence PA-induced lipotoxicity. When we pretreated mice with DUB, an and role of SOCE in lipotoxicity by PA. Since ER luminal protein inhibitor of mPT, hepatocyte death detected by TUNEL staining STIM1 oligomerizes and recruits plasma membrane SOCE channel or elevated serum ALT/AST levels was signiﬁcantly reduced (Fig. 2+ 2+ ORAI1 to activate Ca entry when ER Ca stores are reduced 5A, B), indicating the role of mPT in lipotoxicity in vivo. [33], we studied expression pattern of STIM1 after PA treatment Mitochondrial ROS in the liver tissue of mice treated with 2+ 2+ that reduced ER Ca store due to ER→ lysosome Ca reﬁlling. In LPS + ethyl palmitate was also reduced by DUB (Fig. 5C). These control-treated cells, neither STIM1 oligomerization nor co- results showing amelioration of PA-induced hepatocyte death localization between STIM1 and ORAI1 was observed (Fig. 4A). In in vivo by dantrolene or DUB, suggestthatlipotoxicityof 2+ contrast, both STIM1 oligomerization and co-localization between hepatocytes in vivo can be mediated by Ca -mediated mPT 2+ STIM1 and ORAI1 were well observed after PA treatment (Fig. 4A), and indicate the role of lysosomal Ca release supported by 2+ 2+ supporting that reduced ER Ca content after PA treatment ER→ lysosome Ca reﬁlling and SOCE in mPT-induced lipo- induced SOCE activation. To validate the role of SOCE in toxicity observed in vitro. 2+ intracellular Ca ﬂux, we studied the effect of BTP2, a blocker 2+ of SOCE [35, 36] on [Ca ] . Indeed, in the presence of BTP2, ER 2+ [Ca ] was signiﬁcantly reduced by PA even without removal of DISCUSSION ER 2+ extracellular Ca . In contrast, without BTP2, PA-induced reduction It is well established that PA, as an effector of metabolic stress 2+ 2+ of [Ca ] was not observed in the presence of extracellular Ca in vitro, can induce stress in diverse organelles such as ER and ER (Fig. 4B), suggesting the occurrence of SOCE after PA treatment mitochondria [2, 4, 8, 9]. However, recent investigation showed 2+ 2+ likely due to ER→ lysosome Ca reﬁlling and decreased [Ca ] that PA can induce stress or dysfunction of lysosome as well ER 2+ 2+ unrelated to SERCA inhibition. Furthermore, the increase of [Ca ] [10–13], such as elevated pH or reduced Ca content due to Cell Death Discovery (2023) 9:100 S.-J. Oh et al. 2+ Fig. 3 Role of ER→ lysosome Ca reﬁlling in lipotoxicity. A After treatment of GEM-CEPIA1er-transfected HepG2 cells with PA for 6 h in a 2+ 2+ Ca -free KRBB (right) or a full medium (left), [Ca ] was determined by confocal microscopy. B After treatment of OGBD-labeled cells with ER PA for 6 h, cells were incubated in a fresh medium without PA in the presence or absence of 10 μM dantrolene (Dan), 10 μM TPEN or 3 μM 2+ Xestospongin C (Xesto C). Recovery of [Ca ] after removal of PA was determined by confocal microscopy (lower). Representative Lys 2+ ﬂuorescence images are shown (upper). Scale bar, 20 μm. C After the same treatment as in B, [Ca ] was determined by radiometric analysis after Fura-2 loading. D After the same treatment as in (B), cell death was evaluated by SYTOX Green staining. E After treatment of GEM- 2+ 2+ CEPIA1er transfected cells loaded with OGBD with PA for 6 h and washout, recovery of [Ca ] and changes of [Ca ] were monitored Lys ER 2+ simultaneously in the absence of extracellular Ca . Data are expressed as the means ± SD of three independent experiments. Statistical comparisons were performed using two-tailed Student’s t-test (A) or one-way ANOVA with Tukey’s multiple comparison test (B–D). (*P < 0.05; **P < 0.01; ***P < 0.001; ns not signiﬁcant). 2+ 2+ 2+ release of Ca from lysosome. Increased cytosolic Ca can channel responsible for PA-induced lysosomal Ca release is not 2+ mediate diverse beneﬁcial or harmful effects on cells. Since Ca is clear. Previous papers have reported the role of TRPML1 or TRPM2 2+ 2+ a well-known inducer of mPT [14, 15], we hypothesized that channel in Ca -orZn -mediated cell death [39–41]. We have 2+ lysosomal Ca release might contribute to cell death after also studied the effect of inhibitors of TRPML1 or TRPM2. However, 2+ treatment with PA or lipotoxicity. Indeed, we observed that Ca ML-SI1, ML-SI3 or N-(p-amylcinnamoyl)anthranilic acid inhibiting could be released from lysosome after PA treatment, which is TRPML1, TRPML1/2/3 and TRPM2, respectively [42–44], did not 2+ likely due to activation of lysosomal Ca exit channel by inhibit PA-induced lipotoxic death of HepG2 cells (Oh S-J et al., 2+ 2+ mitochondrial ROS. After release of lysosomal Ca , [Ca ] was unpublished results). Furthermore, Ned-19, an inhibitor of TPC1/2 increased, which imposed mPT and subsequent death. Cell death which has been reported to be associated with ischemia- 2+ due to mPT caused by PA-induced lysosomal Ca release was reperfusion injury [45], was also without effect (Oh S-J et al., 2+ inhibited by mPT inhibitors such as olesoxime or DUB, showing unpublished results). Besides such lysosomal Ca exit channels, 2+ the role of lysosomal Ca release and consequent mPT in other members of the TRPM family or those belonging to different 2+ lipotoxicity, which is consistent with previous papers reporting families might play a role in Ca -mediated lipotoxic cell death, 2+ mPT induction by PA [2, 38]. Identity of lysosomal Ca exit which could be a subject for further studies. It remains to be Cell Death Discovery (2023) 9:100 S.-J. Oh et al. Fig. 4 SOCE in lipotoxicity. A After treatment of HepG2 cells transfected with YFP-STIM1 and mCherry-Orai1 with PA for 6 h, STIM1 aggregation and its co-localization with ORAI1 were examined at the middle and bottom of the cells by confocal microscopy (left). Scale bar, 10 μm. Pearson’s correlation coefﬁcient was calculated as an index of co-localization of YFP-STIM1 and mCherry-Orai1 (right). B After treatment of 2+ GEM-CEPIA1er-transfected cells with PA in the presence or absence of 10 μM BTP2 for 6 h, [Ca ] was determined by confocal microscopy. ER 2+ C, D After the same treatment as in (B), [Ca ] was determined by confocal microscopy after Fluo-3 AM loading (C) or ratiometric analysis after Fura-2 loading (D). E, F After treatment of cells with PA in the presence or absence of 10 μM BTP2 (E) or 2 mM EGTA (F) for 24 h, cell death was evaluated using SYTOX Green. Data are expressed as the means ± SD of three independent experiments. Statistical comparisons were performed using two-tailed Student’s t-test (A) or one-way ANOVA with Tukey’s multiple comparison test (B–F). (*P < 0.05; **P < 0.01; ***P < 0.001; ns not signiﬁcant). Cell Death Discovery (2023) 9:100 S.-J. Oh et al. Fig. 5 In vivo lipotoxicity. A After injection of ethyl palmitate and LPS to C57BL/6 mice with or without pretreatment with dantrolene or DUB, cell death was evaluated by TUNEL staining of hepatic sections (right). Representative TUNEL staining is presented (left) (n= 8–9/group). Insets were magniﬁed. Scale bar, 200 μm. B In serum from mice of (A), ALT and AST levels were determined using a blood chemistry analyzer (n= 8–9/group). C In frozen hepatic sections from the mice of (A), mitochondrial ROS accumulation was determined by MitoSOX staining (n= 5–6/group) (right). Representative ﬂuorescence images are shown (left). Scale bar, 20 μm. Data are presented as the means ± SD. All statistical comparisons were performed using one-way ANOVA with Tukey’s multiple comparison test. (*P < 0.05; **P <0.01; ***P < 0.001; ns not signiﬁcant). Cell Death Discovery (2023) 9:100 S.-J. Oh et al. 2+ clariﬁed which lysosomal Ca channel is involved in lipotoxicity MATERIALS AND METHODS 2+ due to lysosomal Ca -mediated mPT. Reagents 2+ Reagents in this study were purchased from following sources: Oregon It has been demonstrated that ER→ lysosome Ca ﬂux occurs 2+ 2+ Green-488 BAPTA-1 dextran (OGBD, O6789), Fluo-3 AM (F23915), when lysosomal Ca content is lowered due to lysosomal Ca TM MitoSOX Red (M36008), SYTOX Green (S7020), BAPTA-AM (O6807), release by mitochondrial stressors [12, 31]. Such reﬁlling of lysosomal 2+ 2+ 2+ calcein-AM (C1430), Fura-2 (F1221) from Thermo Fisher Scientiﬁc; palmitate Ca pool from ER Ca pool occurring when lysosomal Ca (PA, P0500), decylubiquinone (DUB, D7911), bovine serum albumin (BSA, content is lowered, is likely due to small lysosomal volume A9418), cobalt chloride (CoCl , 232696), ethylene glycol-bis (2-aminoethy- 2+ 2+ accommodating Ca which is 10% of ER Ca volume [30]. Thus, lether)-N,N,N′,N′-tetraacetic acid (EGTA, 03777), MitoTEMPO (SML0737), CA- 2+ 2+ 2+ while [Ca ] is comparable to [Ca ] , lysosomal Ca content Lys ER 074Me (205531) from Sigma-Aldrich; Gly-Phe β-naphthylamide (GPN, 2+ might not be sufﬁcient for progression of events requiring Ca ﬂux. ab145914) from Abcam; BTP2 (S8380) from Selleckchem; CytoTox 96® 2+ 2+ TM Besides ER Ca pool, another source of lysosomal Ca could be Non-Radioactive Cytotoxicity Assay (G1780) from Promega Corporation. 2+ endocytic pathway. However, most of endocytic Ca has been reported to be dissipated before reaching lysosome [46], supporting Cell culture and treatment 2+ 2+ the importance of ER Ca as a dominant source of lysosomal Ca HepG2 cells obtained from Korean Cell Line Bank (KCLB) were grown in DMEM 2+ pool. The role of ER→ lysosome Ca ﬂux in diverse pathological and medium (Welgene, LM-001-05)-1% penicillin–streptomycin–amphotericin B physiological conditions could be an intriguing topic to be explored mixture (Lonza, 17-745E) supplemented with 10% fetal bovine serum (Corning, 35-010-CV). Cells were tested for mycoplasma contamination using in future studies. When we studied whether similar phenomenon 2+ a Mycoplasma PCR Detection Kit (e-MycoTM, 25236, iNtRON Biotechnology). occurs after PA treatment of HepG2 cells, ER→ lysosome Ca PA stock solution was prepared by dissolving palmitate in 70% ethanol and reﬁlling could be clearly demonstrated by simultaneous monitoring 2+ 2+ 2+ heating at 56 °C. PA stock solution was diluted in 2% fatty acid-free BSA- of [Ca ] and [Ca ] during incubation in a Ca -free medium ER Lys DMEM before treatment. For in vitro treatment, following concentrations were 2+ ensuring recovery of [Ca ] after removal of PA. Furthermore, TM Lys employed: OGBD, 100 μg/ml;Fluo-3AM, 5 μM; MitoSOX Red, 5 μM; SYTOX 2+ blockade of ER→ lysosome Ca reﬁlling with dantrolene, an Green Nucleic Acid Stain, 1 μM; BAPTA-AM, 30 μM; calcein-AM, 1 μM; palmitic 2+ 2+ inhibitor of RyR Ca exit channel on ER or chelation of ER Ca acid (PA), 500 μM; decylubiquinone (DUB), 200 μM; cobalt chloride (CoCl ), by TPEN could inhibit lipotoxicity, which indicates that intracellular 2mM; Gly-Phe β-naphthylamide (GPN), 200 μM; BTP2, 10 μM; MitoTEMPO, 2+ 2+ Ca ﬂux from ER occurs to sustain lysosomal Ca release, resulting 10 μM; EGTA, 2 mM; CA-074Me, 10 μM. 2+ 2+ in cell death. Ca ﬂux from ER to lysosome, in turn, induced ER Ca emptying which activated SOCE. Occurrence of SOCE after PA SYTOX Green nucleic acid staining 2+ TM treatment of HepG2 cells was evidenced by no decrease of [Ca ] ER Cell death was evaluated using SYTOX Green Nucleic Acid Stain kit 2+ in cells treated with PA without removal of extracellular Ca , (Thermo Fisher Scientiﬁc, S7020). Brieﬂy, HepG2 cells were seeded in a 24- 2+ reappearance of the decrease of [Ca ] by BTP2 even in the well plate. After 24 h, cells were treated with PA with or without indicated ER 2+ compound for 24 h. Cells were then incubated with SYTOX Green (1 μM) presence of extracellular Ca and STIM1 aggregation co-localized for 30 min at 37 °C. SYTOX Green ﬂuorescence was measured by ﬂow with ORAI1. Functional role of SOCE after treatment with PA was cytometry using BD FACSVerse and FACSCanto II (BD Biosciences, San Jose, demonstrated by inhibition of lipotoxicity by BTP2 or extracellular 2+ CA, USA). Data analysis was performed using FlowJo software (10.8, FlowJo, Ca chelation with EGTA. Furthermore, occurrence of SOCE and no LLC, BD Biosciences). 2+ decrease of [Ca ] in cells treated with PA without removal of ER 2+ 2+ extracellular Ca supports that increase of [Ca ] after PA treatment 2+ LDH release assay is not due to direct release of Ca from ER through SERCA inhibition, TM 2+ Cell death was assessed using an LDH release assay kit (Promega sinceincrease[Ca ] due to SERCA inhibition would not be affected 2+ Corporation, G1780). Brieﬂy, HepG2 cells seeded 4 × 10 well in a 96-well by extracellular Ca [32]. While SERCA inhibition is an important plate were treated with PA with or without indicated compound for 24 h. component of ER stress associated with lipid overload or obesity Culture supernatant was collected and analyzed according to the in vivo inducing altered membrane phospholipid composition [47], manufacturer’s protocol. SERCA might not be important in acute lipotoxicity of HepG2 cells in vitro. However, we do not eliminate the possibility that ER stress Transfection and plasmids might contribute to lipotoxicity in vitro. In fact, we have observed Cells were transfected with plasmids such as GCaMP3-ML1, GEM-CEPIA1er, that CHOP, an important player in ER stress-induced cell death, can TM YFP-STIM1, 3xFLAG-mCherry Red-Orai1/P3XFLAG7.1 using PolyJet In Vitro be activated by mitochondrial ROS produced after PA treatment [48]. ® DNA Transfection Reagent (SigmaGen Laboratories, SL100688), according 2+ In our experiments to investigate the role of increased Ca and to the manufacturer’s protocol. mPT in lipotoxicity in vivo, we observed that DUB inhibiting mPT 2+ and dantrolene abrogating [Ca ] increase and cell death in vitro 2+ Measurement of cytosolic, ER and lysosome Ca contents by PA could inhibit lipotoxic death of hepatocytes in vivo. 2+ To measure [Ca ] , HepG2 cells grown on a chambered coverglass Lys Reduction of mitochondrial ROS by dantrolene or DUB on (Thermo Fisher Scientiﬁc, 155383) were loaded with 100 μg/ml OGBD, an 2+ mitochondrial ROS could be due to a feed-forward response indicator of lysosomal luminal Ca for 16 h. After incubation in a fresh 2+ leading to further ROS release following Ca -mediated mPT media for 4 h, cells were treated with BSA or 500 μM PA for 6 h. After 2+ [49, 50] which was inhibited by dantrolene or DUB. Dantrolene is a washing with Ca -free HEPES-buffered saline (HBS), ﬂuorescence was measured using an LSM780 confocal microscope (Zeiss, Oberkochen, candidate for therapeutic drug against malignant hyperthermia or Germany) and quantiﬁed using ImageJ software. Alzheimer’s disease [51, 52]. DUB has also been studied as a 2+ To determine [Ca ] by confocal microscopy, HepG2 cells grown on potential therapeutic agent against Friedreich’s ataxia [53]or chambered coverglass were loaded with 5 μM Fluo-3 AM for 30 min, an tumor-induced angiogenesis [54]. Our results suggest the 2+ indicator of cytosolic Ca . After treatment with 500 μM PA for 6 h, cells possibility that dantrolene or DUB could be considered candidates 2+ were washed with Ca -free PBS twice, and ﬂuorescence was recorded for drug agents against diseases characterized by lipotoxicity such using an LSM780 confocal microscope. Fluorescence intensity was 2+ as nonalcoholic steatohepatitis or cardiomyopathy. While we have quantiﬁed using ImageJ software. For ratiometric [Ca ] measurement, 2+ shown the contribution of mPT induced by lysosomal Ca release cells were loaded with 2 μM of the acetoxymethyl ester form of Fura-2 in 2+ as a mechanism of lipotoxicity in vitro and in vivo, other previously DMEM at 37 °C for 30 min and then washed with a basal Ca solution (145 mM NaCl, 5 mM KCl, 3 mM MgCl , 10 mM glucose, 1 mM EGTA, 20 mM reported mechanisms of lipotoxicity such as ER or mitochondrial 2 HEPES, pH 7.4). Measurements were conducted using MetaFluor on an stress and generation of ceramide or lysophosphatidylcholine Axio Observer A1 (Zeiss) equipped with a 150 W xenon lamp Polychrome V [1–4] could also play an important role depending on the cellular (Till Photonics, Bloaaom Drive Victor, NY, USA), a CoolSNAP-Hq2 digital context or environmental condition, and optimal strategy against camera (Photometrics, Tucson, AZ, USA), and a Fura-2 ﬁlter set. lipotoxicity could be different accordingly. Cell Death Discovery (2023) 9:100 S.-J. Oh et al. Fluorescence at 340/380 nm was measured in phenol red-free medium, Humane Care and Use of Laboratory Animals. The protocol was 2+ and converted to [Ca ] using the following equation [23]. approved by the Institutional Animal Care and Use Committee (IACUC) of the Department of Laboratory Animal Resources of Soonchunhyang 2þ Ca ¼ K ´ ½ðÞ R R =ðÞ R R ´ F =F d min max minð380Þ maxð380Þ Institute of Medi-bio Science. Ethyl palmitate (Tokyo Chemical Industry, P0003) was dissolved in water with 4.8% lecithin (FUJIFILM Wako Pure where K = Fura-2 dissociation constant (224 nM at 37 °C), F = the d min(380) Chemical Corporation, 120-00832) and 10% glycerol (Sigma-Aldrich, 2+ 380 nm ﬂuorescence in the absence of Ca , F = 380 nm ﬂuores- max(380) G2025) to make a mixture containing ethyl palmitate at a concentration 2+ cence with saturating Ca , R = 340/380 nm ﬂuorescence ratio, R = max of 300 mM. LPS (Sigma-Aldrich, E. coli O55:B5) was dissolved in PBS, and 2+ 340/380 nm ratio with saturating Ca , and R = 340/380 nm ratio in the min 0.025 mg/kg LPS was injected intraperitoneally into mice. Dantrolene 2+ absence of Ca . andDUB were dissolvedinDMSO anddiluted with PBS. For invivo 2+ 2+ To determine ER Ca contents ([Ca ] ), HepG2 cells were grown on ER administration, dantrolene (10 mg/kg), DUB (5 mg/kg) or DMSO solution chambered coverglass and transfected with a GEM-CEPIA1er plasmid, a diluted in PBS was injected into mice that were fasted for 24 h. After 1 h, 2+ ratiometric ﬂuorescent indicator of ER Ca . After 24 h, cells were 300 mM ethyl palmitate or vehicle was administered, followed by LPS 2+ treated with BSA or 500 μM PA for 6 h, and then washed with Ca -free injection 1 h later. Blood and liver tissue samples were obtained KRBB (Sigma-Aldrich, K4002). GEM-CEPIA1er ﬂuorescence was measured 24 h later. using an LSM780 confocal microscope at an excitation wavelength of 405 nm and an emission wavelength of 466 or 520 nm. Fluorescence 2+ ratio F466/F520 was calculated as an index of [Ca ] [32]. Blood chemistry ER Serum ALT and AST levels were measured using a Fuji Dri-Chem NX500i chemistry Analyzer (Fujiﬁlm, Tokyo, Japan). 2+ GCaMP3-ML1 Ca imaging 2+ To detect perilysosomal Ca release, HepG2 cells were transfected with a 2+ 2+ GCaMP3-ML1 Ca probe, a lysosome-targeted genetically-encoded Ca TUNEL staining sensor. Twenty-four h after transfection, cells were treated with BSA or Parafﬁn-embedded liver tissue blocks were prepared by ﬁxing in 10% 2+ 500 μM PA for 6 h. Perilysosomal Ca release was recorded by monitoring neutral buffered formalin (Sigma, HT501128). TUNEL staining was 2+ GCaMP3 ﬂuorescence intensity at 470 nm in a basal Ca solution (145 mM conducted using In Situ Cell Death Detection kit (Roche, 11684817910) NaCl, 5 mM KCl, 3 mM MgCl , 10 mM glucose, 1 mM EGTA, 20 mM HEPES, according to the manufacturer’s instructions. Cell death index was pH 7.4) using a Zeiss LSM780 confocal microscope. GPN, a lysosomotropic expressed as the number of TUNEL-positive cells per ﬁeld counted from 2+ agent, of 200 μM was added to evoke lysosomal Ca release. GCaMP3- more than 20 ﬁelds randomly selected (×200). ML1 ﬂuorescence was calculated as a change GCaMP3 ﬂuorescence (ΔF) over baseline ﬂuorescence (F ). Statistical analysis Data are presented as means ± SD of three independent experiments. Two- STIM1 and ORAI1 co-localization tailed Student’s t-test was used to compare values between two groups. After transfection with YFP-STIM1 and 3xFLAG-mCherry Red-Orai1/ One-way ANOVA with Tukey’s test was used to compare values between P3XFLAG7.1 (provided by Yuan J through Cha S-G) plasmids for 48 h, multiple groups. Data were considered signiﬁcant when P < 0.05. Data HepG2 cells were treated with BSA or 500 μM PA for 6 h. Cells were then analysis was performed using GraphPad Prism 8 software (GraphPad ﬁxed with 4% paraformaldehyde (PFA) at room temperature for 10 min. Software, La Jolla, CA, USA). Fluorescence images were acquired with an LSM780 confocal microscope, and the co-localization between STIM1 and ORAI1 was examined by calculating Pearson’s correlation coefﬁcient using ZEN software (Carl Zeiss DATA AVAILABILITY Microscopy GmbH, Jena, Germany). The data generated or analyzed during the current study and materials are available from the corresponding author without imposed restriction on reasonable request. Measurement of mitochondrial ROS TM Mitochondrial ROS was measured using MitoSOX Red. Brieﬂy, HepG2 REFERENCES cells were pretreated with 10 μM MitoTEMPO for 1 h, and then treated with TM 500 μM PA for 24 h. After incubation with 5 μM MitoSOX Red at 37 °C for 1. Han MS, Park SY, Shinzawa K, Kim S, Chung KW, Lee JH, et al. Lysopho- 15 min, ﬂuorescence was measured by ﬂow cytometry using BD FACSVerse sphatidylcholine as a death effector in lipoapoptosis of hepatocytes. J Lipid Res. or FACSCanto II. Data were analyzed using FlowJo software. 2008;49:84–97. To determine mitochondrial ROS accumulation in vivo, frozen sections 2. Koshkin V, Dai FF, Robson-Doucette CA, Chan CB, Wheeler MB. Limited mito- TM of the liver tissue were incubated with MitoSOX Red at 37 °C for 15 min, chondrial permeabilization is an early manifestation of palmitate-induced lipo- and then washed with PBS. Fluorescence images were observed with an toxicity in pancreatic beta-cells. J Biol Chem. 2008;283:7936–48. LSM710 confocal microscope and ﬂuorescence intensity was quantiﬁed 3. Shimabukuro M, Wang MY, Zhou YT, Newgard CB, Unger RH. Protection against using ImageJ software (NIH, Bethesda, MD, USA). lipoapoptosis of b cells through leptin-dependent maintenance of Bcl-2 expression. Proc Natl Acad Sci USA. 1998;95:9558–61. 4. Sommerweiss D, Gorski T, Richter S, Garten A, Kiess W. Oleate rescues INS-1E mPTP opening 2+ β-cells from palmitate-induced apoptosis by preventing activation of the unfol- mPTP opening was assessed by Co quenching of calcein-AM ﬂuores- ded protein response. Biochem Biophys Res Com. 2013;441:770–6. cence. Cells were treated with PA (500 μM) with or without DUB (200 μM) 5. Katsoulieris E, Mabley JG, Samai M, Green IC, Chatterjee PK. alpha-Linolenic acid or olesoxime (100 μM) for 24 h. Cells were then loaded with calcein-AM protects renal cells against palmitic acid lipotoxicity via inhibition of endoplasmic (1 μM) and CoCl (2 mM) at 37 °C for 15 min. Calcein ﬂuorescence was reticulum stress. Eur J Pharmacol. 2009;623:108–12. measured by ﬂow cytometry employing BD FACSVerse and FACSCanto II 6. Khan MJ, Alam MR, Waldeck-Weiermair M, Karsten F, Groschner L, Riederer M, and analyzed using FlowJo software. et al. Inhibition of autophagy rescues palmitic acid-induced necroptosis of endothelial cells. J Biol Chem. 2012;287:21110–20. Mitochondrial membrane potential 7. Shimabukuro M, Zhou YT, Levi M, Unger RH. Fatty acid-induced b cell apoptosis: a Mitochondrial membrane potential was determined using tetramethylrhoda- link between obesity and diabetes. Proc Natl Acad Sci USA. 1998;95:2498–502. mine ethyl ester perchlorate (TMRE). After treatment of HepG2 cells with PA 8. Sparagna GC, Hickson-Bick DL, Buja LM, McMillin JB. A metabolic role for mito- for 24 h, cells were incubated with 100 nM TMRE at 37 °C for 30 min, and chondria in palmitate-induced cardiac myocyte apoptosis. Am J Physiol. ﬂuorescence was measured using LSM710 confocal microscope. Fluorescence 2000;279:H2124–2132. intensity was quantiﬁed using ImageJ software. 9. Xu S, Nam SM, Kim JH, Das R, Choi SK, Nguyen TT, et al. Palmitate induces ER calcium depletion and apoptosis in mouse podocytes subsequent to mito- chondrial oxidative stress. Cell Death Dis. 2015;6:e1976. Animals 10. Karasawa T, Kawashima A, Usui-Kawanishi F, Watanabe S, Kimura H, Kamata R, Eight-week-old C57BL/6N male mice were purchased from Orient Bio et al. Saturated fatty acids undergo intracellular crystallization and activate the (Seongnam, Korea). All experiments using mice were performed in NLRP3 inﬂammasome in macrophages. Arterioscler Thromb Vasc Biol. accordance with the guidelines of the Public Health Service Policy in 2018;38:744–56. Cell Death Discovery (2023) 9:100 S.-J. Oh et al. 11. Kim J, Kim SH, Kang H, Lee S, Park S-Y, Cho Y, et al. TFEB-GDF15 axis protects 40. Du W, Gu M, Hu M, Pinchi P, Chen W, Ryan M, et al. Lysosomal Zn 2+ release against obesity and insulin resistance as a lysosomal stress response. Nature triggers rapid, mitochondria-mediated, non-apoptotic cell death in metastatic Metabolism. 2021;3:410–27. melanoma. Cell Rep. 2021;37:109848. 12. Park K, Lim H, Kim J, Hwang H, Lee YS, Bae SH, et al. Essential role of lysosomal 41. Zaibi N, Li P, Xu S-Z. Protective effects of dapagliﬂozin against oxidative stress- Ca2+-mediated TFEB activation in mitophagy and functional adaptation of induced cell injury in human proximal tubular cells. PLoS ONE. 2021;16:e0247234. pancreatic β-cells to metabolic stress. Nat Commun. 2022;13:1300. 42. Kraft R, Grimm C, Frenzel H, Harteneck C. Inhibition of TRPM2 cation channels by 13. Trudeau KM, Colby AH, Zeng J, Las G, Feng JH, Grinstaff MW, et al. Lysosome N-(p-amylcinnamoyl)anthranilic acid. Br J Pharma. 2006;148:264–73. acidiﬁcation by photoactivated nanoparticles restores autophagy under lipo- 43. Samie M, Wang X, Zhang X, Goschka A, Li X, Cheng X, et al. A TRP channel in the toxicity. J Cell Biol. 2016;214:25–34. lysosome regulates large particle phagocytosis via focal exocytosis. Dev Cell. 14. Fontaine E, Ichas F, Bernardi P. A ubiquinone-binding site regulates the mito- 2013;26:511–24. chondrial permeability transition pore. J Biol Chem. 1998;273:25734–40. 44. Sun J, Liu Y, Hao X, Lin W, Su W, Chiang E, et al. LAMTOR1 inhibition of TRPML1- 15. Nakagawa T, Shimizu S, Watanabe T, Yamaguchi O, Otsu K, Yamagata H, et al. dependent lysosomal calcium release regulates dendritic lysosome trafﬁcking Cyclophilin D-dependent mitochondrial permeability transition regulates some and hippocampal neuronal function. EMBO J. 2022;41:e108119. necrotic but not apoptotic cell death. Nature. 2005;434:652–8. 45. Simon JS, Vrellaku B, Monterisi S, Chu SM, Rawlings N, Lomas O, et al. Oxidation of 16. Schönfeld P, Wojtczak L. Fatty acids as modulators of the cellular production of protein kinase A regulatory subunit PKARIα porotects against myocardial reactive oxygen species. Free Radic Biol Med. 2007;45:231–41. ischemia-reperfusion injury byinhibiting lysosomal-triggered calcium release. 17. Zhang X, Cheng X, Yu L, Yang J, Calvo R, Patnaik S, et al. MCOLN1 is a ROS sensor Circulation. 2020;143:449–65. in lysosomes that regulates autophagy. Nat Commun. 2016;7:12109. 46. Gerasimenko JV, Tepikin AV, Petersen OH, Gerasimenko OV. Calcium uptake via 18. Chang I, Cho N, Kim S, Kim JY, Kim E, Woo JE, et al. Role of calcium in pancreatic endocytosis with rapid release from acidifying endosomes. Curr Biol. 1998;8:1335–8. islet cell death by IFN-gamma/TNF-alpha. J Immunol. 2004;172:7008–14. 47. Fu S, Yang L, Li P, Hofmann O, Dicker L, Hide W, et al. Aberrant lipid metabolism 19. Maher P, van Leyen K, Dey PN, Honrath B, Dolga A, Methner A. The role of Ca 2+ disrupts calcium homeostasis causing liver endoplasmic reticulum stress in in cell death caused by oxidative glutamate toxicity and ferroptosis. Cell Calcium. obesity. Nature. 2011;473:528–31. 2018;70:47–55. 48. Ly LD, Ly DD, Nguyen NT, Kim J-H, Yoo H, Chung J, et al. Mitochondrial Ca2+ 20. Zhivotovsky B, Orrenius S. Calcium and cell death mechanisms: a perspective uptake relieves palmitate-induced cytosolic Ca2+ overload in MIN6 cells. Mol from the cell death community. Cell Calcium. 2011;50:211–21. Cells. 2020;43:66–75. 21. Hunnicutt JH, Hardy RW, Williford J, McDonald JM. Saturated fatty acid-induced 49. Zorov DB, Filburn CR, Klotz LO, Zweier JL, Sollott SJ. Reactive oxygen species insulin resistance in rat adipocytes. Diabetes. 1994;43:540–5. (ROS)-induced ROS release: a new phenomenon accompanying induction of the 22. Shen D, Wang X, Li X, Zhang X, Yao Z, Dibble S, et al. Lipid storage disorders block mitochondrial permeability transition in cardiac myocytes. J Exp Med. lysosomal trafﬁcking by inhibiting a TRP channel and lysosomal calcium release. 2000;192:1001–14. Nat Commun. 2012;3:731. 50. Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial reactive oxygen species (ROS) 23. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with and ROS-induced ROS release. Physiol Rev. 2014;94:909–50. greatly improved ﬂuorescence properties. J Biol Chem. 1985;260:3440–50. 51. Karagas NE, Venkatachalam K. Roles for the endoplasmic reticulum in regulation 24. Li Z, Berk M, McIntyre TM, Gores GJ, Feldstein AE. The lysosomal-mitochondrial of neuronal calcium homeostasis. Cells. 2019;8:1232. axis in free fatty acid-induced hepatic lipotoxicity. Hepatology. 2008;47:1495–503. 52. Kim KSM, Kriss RS, Tautz TJ. Malignant hyperthermia: a clinical review. Adv 25. Rial E, Rodríguez-Sánchez L, Gallardo-Vara E, Zaragoza P, Moyano E, González- Anesth. 2019;37:35–51. Barroso MM. Lipotoxicity, fatty acid uncoupling and mitochondrial carrier func- 53. Jauslin ML, Meier T, Smith RA, Murphy MP. Mitochondria-targeted antioxidants tion. Biochim Biophys Acta. 2008;1979:800–6. protect Friedreich Ataxia ﬁbroblasts from endogenous oxidative stress more 26. Petronilli V, Miotto G, Canton M, Colonna R, Bernardi P, Di Lisa F. Imaging the effectively than untargeted antioxidants. FASEB J. 2003;17:1972–4. mitochondrial permeability transition pore in intact cells. Biofactors. 1998;8:263–72. 54. Cao J, Liu X, Yang Y, Wei B, Li Q, Mao G, et al. Decylubiquinone suppresses breast 27. Bradham CA, Qian T, Streetz K, Trautwein C, Brenner DA, Lemasters JJ. The cancer growth and metastasis by inhibiting angiogenesis via the ROS/p53/ mitochondrial permeability transition is required for tumor necrosis factor alpha- BAI1 signaling pathway. Angiogenesis. 2020;23:325–38. mediated apoptosis and cytochrome c release. Mol Cell Biol. 1998;18:6353–64. 28. Halestrap AP, Kerr PM, Javadov S, Woodﬁeld KY. Elucidating the molecular mechanism of the permeability transition pore and its role in reperfusion injury of the heart. Biochim Biophys Acta. 1998;1366:79–94. AUTHOR CONTRIBUTIONS 29. Bordet T, Berna P, Abitbol J-L, Pruss RM. Olesoxime (TRO19622): a novel M-SL and KYH conceived the experiments. S-JO and YH conducted the experiments. mitochondrial-targeted neuroprotective compound. Pharmaceuticals. 2010;3:345–68. M-SL, KYH, S-JO and YH wrote the manuscript. 30. Penny CJ, Kilpatrick BS, Han JM, Sneyd J, Patel S. A computational model of lysosome-ER Ca2+ microdomains. J Cell Sci. 2014;127:2934–43. 31. Garrity AG, Wang W, COllier CMD, Levery SA, Gao Q, Xu H. The endoplasmic reticulum, not the pH gradient, drives calcium reﬁlling of lysosomes. eLife. FUNDING 2016;5:e15887. This study was supported by a National Research Foundation of Korea (NRF) grant 32. Suzuki J, Kanemaru K, Ishii K, Ohkura M, Lino M. Imaging intraorganellar Ca2+ at funded by the Korea government (MSIT) (NRF-2019R1A2C3002924) and by the subcellular resolution using CEPIA. Nat Commun. 2014;5:4153. Bio&Medical Technology Development Program (2017M3A9G7073521). M-S Lee is 33. Derler I, Jardin I, Romanin C. Molecular mechanisms of STIM/Orai communication. the recipient of Korea Drug Development Fund from the Ministry of Science & ICT, Am J Physiol. 2016;310:C643–662. Ministry of Trade, Industry & Energy and Ministry of Health & Welfare (HN22C0278), 34. Hofer AM, Fasolato C, Pozzan T. Capacitative Ca2+ entry is closely linked to the and a grant from the Faculty Research Assistance Program of Soonchunhyang ﬁlling state of internal Ca2+ stores: a study using simultaneous measurements of University (20220346). ICRAC and Intraluminal [Ca2+]. J Cell Biol. 1998;130:325–34. 35. Bogeski I, Al-Ansary D, Qu B, Niemeyer BA, Hoth M, Peinelt C. Pharmacology of ORAI channels as a tool to understand their physiological functions. Expert Rev Clin Pharmacol. 2010;3:291–303. COMPETING INTERESTS 36. Zitt C, Strauss B, Schwarz EC, Spaeth N, Rast G, Hatzelmann A, et al. Potent M-SL in the CEO of LysoTech, Inc. The authors declare no competing interests. inhibition of Ca2+ release-activated Ca2+ channels and T-lymphocyte activation by the pyrazole derivative BTP2. J Biol Chem. 2004;279:12427–37. 37. Ogawa Y, Imajo K, Honda Y, Kessoku T, Tomeno W, Kato S, et al. Palmitate- induced lipotoxicity is crucial for the pathogenesis of nonalcoholic fatty liver ADDITIONAL INFORMATION disease in cooperation with gut-derived endotoxin. Sci Rep. 2018;8:11365. Correspondence and requests for materials should be addressed to Myung-Shik Lee. 38. Srivastava S, Chan C. Hydrogen peroxide and hydroxyl radicals mediate palmitate-induced cytotoxicity to hepatoma cells: relation to mitochondrial Reprints and permission information is available at http://www.nature.com/ permeability transition. Free Radic Res. 2007;41:38–49. reprints 39. Akyuva Y, Nazıroğlu M. Resveratrol attenuates hypoxia-induced neuronal cell death, inﬂammation and mitochondrial oxidative stress by modulation of TRPM2 Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims channel. Sci Rep. 2020;10:6449. in published maps and institutional afﬁliations. Cell Death Discovery (2023) 9:100 S.-J. Oh et al. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http:// creativecommons.org/licenses/by/4.0/. © The Author(s) 2023 Cell Death Discovery (2023) 9:100

Journal

Cell Death Discovery – Springer Journals

Published: Mar 21, 2023

References

Lysophosphatidylcholine as a death effector in lipoapoptosis of hepatocytes

Han, MS; Park, SY; Shinzawa, K; Kim, S; Chung, KW; Lee, JH
Limited mitochondrial permeabilization is an early manifestation of palmitate-induced lipotoxicity in pancreatic beta-cells

Koshkin, V; Dai, FF; Robson-Doucette, CA; Chan, CB; Wheeler, MB
Protection against lipoapoptosis of b cells through leptin-dependent maintenance of Bcl-2 expression

Shimabukuro, M; Wang, MY; Zhou, YT; Newgard, CB; Unger, RH
Oleate rescues INS-1E β-cells from palmitate-induced apoptosis by preventing activation of the unfolded protein response

Sommerweiss, D; Gorski, T; Richter, S; Garten, A; Kiess, W
alpha-Linolenic acid protects renal cells against palmitic acid lipotoxicity via inhibition of endoplasmic reticulum stress

Katsoulieris, E; Mabley, JG; Samai, M; Green, IC; Chatterjee, PK
Inhibition of autophagy rescues palmitic acid-induced necroptosis of endothelial cells

Khan, MJ; Alam, MR; Waldeck-Weiermair, M; Karsten, F; Groschner, L; Riederer, M
Fatty acid-induced b cell apoptosis: a link between obesity and diabetes

Shimabukuro, M; Zhou, YT; Levi, M; Unger, RH
A metabolic role for mitochondria in palmitate-induced cardiac myocyte apoptosis

Sparagna, GC; Hickson-Bick, DL; Buja, LM; McMillin, JB
Palmitate induces ER calcium depletion and apoptosis in mouse podocytes subsequent to mitochondrial oxidative stress

Xu, S; Nam, SM; Kim, JH; Das, R; Choi, SK; Nguyen, TT
Saturated fatty acids undergo intracellular crystallization and activate the NLRP3 inflammasome in macrophages

Karasawa, T; Kawashima, A; Usui-Kawanishi, F; Watanabe, S; Kimura, H; Kamata, R
TFEB-GDF15 axis protects against obesity and insulin resistance as a lysosomal stress response

Kim, J; Kim, SH; Kang, H; Lee, S; Park, S-Y; Cho, Y
Essential role of lysosomal Ca2+-mediated TFEB activation in mitophagy and functional adaptation of pancreatic β-cells to metabolic stress

Park, K; Lim, H; Kim, J; Hwang, H; Lee, YS; Bae, SH
Lysosome acidification by photoactivated nanoparticles restores autophagy under lipotoxicity

Trudeau, KM; Colby, AH; Zeng, J; Las, G; Feng, JH; Grinstaff, MW
A ubiquinone-binding site regulates the mitochondrial permeability transition pore

Fontaine, E; Ichas, F; Bernardi, P
Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death

Nakagawa, T; Shimizu, S; Watanabe, T; Yamaguchi, O; Otsu, K; Yamagata, H
Fatty acids as modulators of the cellular production of reactive oxygen species

Schönfeld, P; Wojtczak, L
MCOLN1 is a ROS sensor in lysosomes that regulates autophagy

Zhang, X; Cheng, X; Yu, L; Yang, J; Calvo, R; Patnaik, S
Role of calcium in pancreatic islet cell death by IFN-gamma/TNF-alpha

Chang, I; Cho, N; Kim, S; Kim, JY; Kim, E; Woo, JE
The role of Ca 2+ in cell death caused by oxidative glutamate toxicity and ferroptosis

Maher, P; van Leyen, K; Dey, PN; Honrath, B; Dolga, A; Methner, A
Calcium and cell death mechanisms: a perspective from the cell death community

Zhivotovsky, B; Orrenius, S
Saturated fatty acid-induced insulin resistance in rat adipocytes

Hunnicutt, JH; Hardy, RW; Williford, J; McDonald, JM
Lipid storage disorders block lysosomal trafficking by inhibiting a TRP channel and lysosomal calcium release

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Lysosomal Ca2+ as a mediator of palmitate-induced lipotoxicity

Lysosomal Ca2+ as a mediator of palmitate-induced lipotoxicity

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Lysosomal Ca2+ as a mediator of palmitate-induced lipotoxicity

Lysosomal Ca2+ as a mediator of palmitate-induced lipotoxicity

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